Methods of treating retinal nerve fiber layer degeneration with monoclonal antibodies against a retinal guidance molecule (RGM) protein

ABSTRACT

The present application describes RGM A binding proteins, particularly monoclonal antibodies, and in particular CDR grafted, humanized versions thereof, which have the ability to bind to RGM A and prevent binding of RGM proteins to RGM A receptor and other RGM A binding proteins, and therefore neutralize the function of RGM A, for use in the treatment of retinal nerve fiber layer (RNFL) degeneration as well as methods of therapeutically or prophylactically treating a mammal against RNFL degeneration.

This application claims priority from U.S. Provisional Application Ser.No. 61/267,446 filed Dec. 8, 2009, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present application describes RGM A binding proteins, particularlymonoclonal antibodies, and in particular CDR grafted, humanized versionsthereof, which have the ability to bind to RGM A and prevent binding ofRGM proteins to RGM A receptor and other RGM A binding proteins, andtherefore neutralize the function of RGM A, for use in the treatment ofretinal nerve fiber layer (RNFL) degeneration as well as methods oftherapeutically or prophylactically treating a mammal against RNFLdegeneration.

BACKGROUND OF THE INVENTION

Axonal regeneration after injury, inflammatory attacks, orneurodegenerative diseases within the mammalian central nervous system(CNS) is almost always impossible; the outcome depends on the balancebetween the intrinsic ability of the nerve fibers in the CNS to re-grow,and the inhibitory factors within the CNS, localized in themicroenvironment of the lesion or damage site, which actively preventthe re-growth, and thus the regeneration of the injured fiber tracts.

It has been established that CNS myelin, generated by oligodendrocytes,and the lesional scar are the most relevant non-permissive structuresfor axonal growth in the early phase of an injury, by causing growthcone collapse and neurite growth inhibition in vitro as well as in vivo,thereby resulting in direct inhibition of axon regrowth. RGM proteins,major inhibitory factors on CNS myelin and scar tissue have beenidentified (Monnier et al., Nature 419: 392-395, 2002; Schwab et al.,Arch. Neurol. 62: 1561-8, 2005a; Schwab et al. Eur. J. Neurosci. 21:1569-76, 2005 b; Hata et al. J. Cell Biol. 173: 47-58, 2006; for reviewssee: Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci. 361:1513-29, 2006; Yamashita et al. Curr. Opin. Neurobiol. 17: 29-34, 2007).RGM proteins are up-regulated at damage or lesion sites in humans dyingfrom brain trauma or ischemic insult, (Schwab et al., Arch. Neurol. 62:1561-8, 2005a) and are up-regulated at lesion sites in rats with spinalcord injury (Schwab et al. Eur. J. Neurosci. 21: 1569-76, 2005 b; Hataet al. J. Cell Biol. 173: 47-58, 2006 for review see: Mueller et al.,Philos. Trans. R. Soc. Lond. B Biol. Sci. 361: 1513-29, 2006; Yamashitaet al. Curr. Opin. Neurobiol. 17: 29-34, 2007). In addition, first datausing clinical samples from Multiple sclerosis patients and healthypersons suggested that human RGM A is up-regulated in cerebrospinalfluid of patients suffering from MS (data not shown).

To evaluate the regeneration-promoting potential of an RGM A-specificpolyclonal antibody, the antibodies were administered in amoderate-to-severe model of spinal cord injury, where approximately 60%of the spinal cord at thoracal level 9/10 was transected. Thehistological examination revealed that such a lesion severed all dorsaland lateral fibers of the corticospinal tract. The RGM A—specificpolyclonal antibody given locally via pump for two weeks inducedlong-distance regeneration of injured nerve fibers (Hata et al., J. CellBiol. 173: 47-58, 2006).

Hundreds of nerve fibers extended past the lesion site and the longestfibers regenerated for more than 10 mm beyond the lesion, whereas noregenerating fibers were found distal to the lesion in controlantibody-treated animals. The functional recovery of the anti-RGM Atreated rats was significantly improved in comparison withcontrol-antibody treated, spinally injured rats, thereby proving thatRGM A is a potent neuroregeneration inhibitor and a valuable target forstimulating recovery in indications characterized by axon damage ornerve fiber injury (Hata et al., J. Cell Biol. 173: 47-58, 2006; Kyotoet al. Brain Res. 1186: 74-86, 2007). In addition neutralising the RGM Aprotein with a function-blocking polyclonal antibody stimulated not onlyregrowth of damaged nerve fibers in the spinally injured rats butenhanced their synapse formation thereby enabling the reformation orrestoration damaged neuronal circuits (Kyoto et al. Brain Res. 1186:74-86, 2007).

The rgm gene family encompasses three different genes, two of them, rgma and b, are expressed in the mammalian CNS originating RGM A and RGM Bproteins, whereas the third member, rgm c, is expressed in the periphery(Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci. 361: 1513-29,2006), where RGM C plays an important role in iron metabolism. In vitro,RGM A inhibits neurite outgrowth by binding to Neogenin, which has beenidentified as an RGM receptor (Rajagopalan et al. Nat Cell Biol.: 6(8),756-62, 2004). Neogenin had first been described as a netrin-bindingprotein (Keino-Masu et al. Cell, 87(2): 175-85, 1996). This is animportant finding because binding of Netrin-1 to Neogenin or to itsclosely related receptor DCC (deleted in colorectal cancer) has beenreported to stimulate rather than to inhibit neurite growth (Braisted etal. J. Neurosci. 20: 5792-801, 2000). Blocking RGM A therefore releasesthe RGM-mediated growth inhibition by enabling Neogenin to bind itsneurite growth-stimulating ligand Netrin. Based on these observations,neutralizing RGM A can be assumed to be superior to neutralizingNeogenin in models of human spinal cord injury. Besides binding of RGM Ato Neogenin and inducing neurite growth inhibition, the binding of RGM Aor B to the bone morphogenetic proteins BMP-2 and BMP-4 could representanother obstacle to successful neuroregeneration and functional recovery(Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci. 361: 1513-29,2006).

The RNFL is the innermost layer of the retina and is principallycomposed of axons from ganglion cell neurons that compose the opticnerve. The axons within the RNFL are not myelinated until they passthrough the lamina cribrosa of the eye. This structural characteristicof the RNFL makes it an ideal tissue to examine neurodegenerativeprocesses within the CNS because RNFL thickness reflects thecontribution of axons without potential structural effects of myelindegeneration (Frohman et al., Arch. Neurol. 65(1): 26-35, 2008) (See,also FIG. 19).

Frisén, L. and Hoyl, W. F. reported for the first time of thinning ofRNFL in patients with Multiple Sclerosis (MS) (Frisén, L. and Hoyl, W.F., Arch. Opthalmol. 92: 91-97, 1974).

In healthy individuals, the RNFL is only about 110-120 μm thick by theage of 15 years and most normal individuals will lose about 0.017% peryear in retinal thickness which equates to about 10-20 μm over 60 years(Kanamori. A. et al., Ophthalmologica 217: 273-278, 2003). Contrary tothis about 75% of patients with MS who had experienced acute opticneuritis (AON) showed 10 to 40 μm RNFL thickness loss within a period ofonly about 3-6 months following the initial inflammatory event. It isalso reported that thinning of the RNFL below a level of about 75 μmcauses a decay of visual function (Costello, et al., Ann. Neurol. 59:963-969, 2006). The potential utility of the RNFL for the purpose ofmodeling neuroprotection in response to MS therapies was suggested byFrohman et al., who also describe optical coherence tomography (OTC) asa reproducible imaging technique allowing measurements of RNFL thickness(Frohman et al., see above; and Frohman et al.; Nature Clinical PracticeNeurology 4: 12, 664-675, 2008).

Degeneration of the RNFL is also observed during the course of numerousother diseases like; diabetic retinopathy, ischemic optic neuropathy,X-chromosome linked retinoschisis, drug-induced optic neuropathy,retinal dystrophy, age-related macula degeneration, eye diseasescharacterized by optic nerve head drusen, eye disease characterized bygenetic determinants of photoreceptor degeneration, autosomal recessivecone-rod dystrophy, mitochondrial disorders with optic neuropathy, i.p.Friedreich's ataxia, Alzheimer's disease, mild cognitive impairment(MCI) Parkinsons's disease, and Prion disease, i.p. Creutzfeld-Jakob,Scrapie, BSE (see also Sakata L M, et al. Clin. Experiment Ophtalmol.2009, 37: 90-99; Morris R W et al. Optometry 2009, 80, 83-100;Kallenbach K. & Frederiksen J. Eur. J. Neurol. 2007, 14: 841-849; Tricket al. J. Neuroophtthalmol. 2006, 26: 284-295; Tantri A. et al. Surv.Ophtalmol. 49: 214-230).

There is a need in the art for a therapeutic approach allowing thedirect treatment of RNFL degeneration. The problem to be solved by thepresent invention was, therefore, to provide means allowing the directtreatment, in particular neuroprotective treatment of RNFL degenerationas observed in a multiplicity of disease conditions.

SUMMARY OF THE INVENTION

An embodiment of the invention relates to neuroprotective treatment ofRNFL degeneration as observed in a multiplicity of disease conditionsusing antibodies capable of binding RGM A.

Another embodiment relates to the use of a monoclonal antibody thatblocks RGM A and prevents the interaction between RGM A and its receptorand/or binding proteins, i.e. Neogenin and BMP-2, BMP-4, for thetreatment of RNFL degeneration.

Another embodiment is a method of treating RNFL degeneration, comprisingthe step of administering to a mammal in need thereof an effectiveamount of a composition comprising antibodies capable of binding RGM A.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the monoclonal antibodies binding to hRGM A in ELISAassay.

FIG. 1B depict the monoclonal antibodies binding to hRGM A expressed inHEK 293 cells.

FIG. 1C depict the monoclonal antibodies binding to rat RGM A expressedin HEK 293 cells.

FIG. 2 shows the full length RGM A binding to Neogenin. MAB 5F9 inhibitsbinding of full length, fc-coupled hRGM A to Neogenin.

FIG. 3 depicts the full length RGM A binding to BMP-4. MAB 5F9 inhibitsbinding of fc-coupled full length hRGM A fragment (47-422) to BMP-4.

FIG. 4 depicts RGM A fragment 0 binding to BMP-4. MAB 5F9 inhibitsbinding of fc-coupled hRGM A fragment 0 (47-168) to BMP-4.

FIG. 5 shows full length RGM A binding to BMP-2. MAB 5F9 inhibitsbinding of fc-coupled full length hRGM A fragment (47-422) to BMP-2.

FIG. 6 is a combination of microphotographs showing mAb5F9neutralization of RGM A fragment in NTera cell neurite outgrowth assay.MAB 5F9 neutralizes the outgrowth inhibitory activity of anfc-conjugated, potent hRGM A inhibitor fragment in neurite growth assayswith human Ntera aggregates. A. Control culture, growth of Ntera neuronson laminin, B. on a laminin-hRGM A fragment (47-168) substrate, C.-E. ona laminin-hRGM A fragment (47-168) substrate in the presence of 0.1μg/ml MAB 5F9 (C.), 1 μg/ml MAB 5F9 (D.), 10 μg/ml MAB 5F9 (E).

FIG. 7 shows the quantitative analysis of NTera 2 assay results. MAB 5F9neutralizes dose-dependently the outgrowth inhibitory activity of anfc-conjugated, potent hRGM A inhibitor fragment (fragment 0, 47-168) inneurite growth assays with human Ntera aggregates.

FIG. 8 shows the quantitative analysis of SH-SY5Y stripe assay. MAB 5F9neutralizes repulsion, induced by stripes consisting of full lengthhuman RGM A of human SH-SY5Y neuronal cells in stripe membrane carpets.In the absence of MAB 5F9 (A) or in the presence of low MABconcentrations SH-SY5Y neurons prefer to avoid the RGM A stripes. Thisbehaviour is reversed by increasing concentrations of the MAB 5F9. (B toD) At the highest MAB concentration (10 μg/ml) (E), SH-SY5Y neurons showa strong preference for the RGM A stripes in comparison with theCollagen I stripes.

FIG. 9 summarizes the quantitative analysis of mABs 5F9 and 8D1 bindingcharacteristics. MABs 5F9 and 8D1 are evaluated in hRGM A—neogenin, hRGMA—BMP-2 and hRGM A—BMP-4 binding assays at different concentrations.

FIG. 10 shows neutralizing activity for hRGM A's chemorepulsive activityof humanized 5F9 antibodies (h5F9.21, h5F9.23, h5F9.25) in an SH-SY5Ychemotaxis assay.

FIG. 11 shows the in vivo neuroregenerative activity of local 5F9application in an animal model of optic nerve injury. Local applicationof MAB 5F9 neutralizes RGM A and stimulates regenerative growth ofdamaged optic nerve axons in a rat animal model of optic nerve crush. Inthe 5F9 treated animals (A), many GAP-43 positive fibers are extendingbeyond the crush site in contrast to the control MAB 8D1 (B), which doesnot bind to rat RGM A.

FIG. 12 A and FIG. 12 B show the quantitative analysis of local 5F9application in an animal model of optic nerve injury. (A) 5F9 but notthe control MAB 8D1 significantly increased the number of regeneratingGAP-43 positive fibers. Significantly more fibers (p<0.05) were observedin animals treated with 5F9 at distances 200 μm, 400 μm and 600 μm andat 1200 μm fibers are only found in 5F9-treated animals but not incontrol animals (B) 5F9 significantly increased the GAP-43 positive areaat the optic nerve lesion site in comparison with the control antibody8D1 and the vehicle control PBS. The area of regenerative growth (GAP-43positive area) was measured using the Axiovision software (Zeiss).

FIG. 13 shows the in vivo neuroregenerative activity of systemic 5F9application in an animal model of optic nerve injury. Animals weretreated with 5F9 at day 0 and day 21 with 2 mg/kg and 10 mg/kg,respectively. Antibody or vehicle were given intraperitoneally orintravenously. Composite images of rat optic nerves. In the 5F9 treatedanimals (A), many GAP-43 positive fibers are extending beyond the crushsite in contrast to control animals treated with PBS (B). The crush siteis located at the left margin and regenerating fibers are stained withan antibody to GAP-43. Many fibers are observed at the upper and lowerrim of the optic nerve in 5F9-treated animals but not in PBS animals.

FIG. 14 A and FIG. 14 B shows the quantitative analysis of systemic 5F9application in an animal model of optic nerve injury.

FIG. 15 shows the in vivo remyelinating activity of systemic 5F9application in an animal model of optic nerve injury. Animals weretreated with 5F9 at day 0 and d21 with 2 mg/kg and 10 mg/kg,respectively. Antibody or vehicle were given intraperitoneally orintravenously. Composite images of rat optic nerves. Myelination isvisualized using an antibody directed against the myelin marker myelinbasic protein MBP. Crush sites ate located in the middle of thecomposite nerves and the area is free in vehicle treated control animals(A and B). In the 5F9 treated animals (C and D), many MBP-positivestructures are observed in the middle area (crush center) of the opticnerves.

FIG. 16 shows the quantitative effect on remyelination of systemic 5F9application in an animal model of optic nerve injury.

FIG. 17 illustrates the superior protective effect of antibody 5F9 onRNFL from eyes of animals with optic nerve crush. A significantly higherdensity of nerve fiber bundles is observed in retinae of animalssystemically treated with 5F9.

FIG. 18 illustrates the influence of antibody 5F9 on intraretinalneurons from eyes of animals with optic nerve crush. A significantlyhigher number of sprouting intraretinal neurons is observed in retinaeof animals systemically treated with the 5F9 antibody.

FIG. 19 illustrates the retinal nerve fiber layer (RNFL), which is thelayer directly adjacent to the vitreous bulb. It is formed by the axonsof retinal ganglion cells. The other layers are the inner plexiformlayer (IPL), where the amacrine and bipolar neurons form synapticcontacts with the retinal ganglion cells. Photoreceptors, horizontalneurons and bipolar neurons makes synapses in the outer plexiform layer(OPL). Photoreceptors (rods and cones) are located in the photoreceptorlayer (PRL). RPE is the retinal pigment epithelium (scheme is fromFrohman et al. Arch. Neurol. 65, 26-35, 2008).

DETAILED DESCRIPTION OF THE INVENTION 1. Definition of Terms

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear, however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms shall include pluralities and plural termsshall include the singular. In this application, the use of “or” means“and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The nomenclatures used in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

As used throughout this specification and the appended claims, thefollowing terms have the following meanings:

The terms “acceptor” and “acceptor antibody” refer to the antibody ornucleic acid sequence providing or encoding at least 80%, at least 85%,at least 90%, at least 95%, at least 98% or 100% of the amino acidsequences of one or more of the framework regions. In some embodiments,the term “acceptor” refers to the antibody amino acid or nucleic acidsequence providing or encoding the constant region(s). In yet anotherembodiment, the term “acceptor” refers to the antibody amino acid ornucleic acid sequence providing or encoding one or more of the frameworkregions and the constant region(s). In a specific embodiment, the term“acceptor” refers to a human antibody amino acid or nucleic acidsequence that provides or encodes at least 80%, particularly, at least85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acidsequences of one or more of the framework regions. In accordance withthis embodiment, an acceptor may contain at least 1, at least 2, atleast 3, least 4, at least 5, or at least 10 amino acid residues thatdoes (do) not occur at one or more specific positions of a humanantibody. An acceptor framework region and/or acceptor constantregion(s) may be, e.g., derived or obtained from a germline antibodygene, a mature antibody gene, a functional antibody (e.g., antibodieswell-known in the art, antibodies in development, or antibodiescommercially available).

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody formats are known in the art. Non-limitingembodiments of which are discussed below. An antibody is said to be“capable of binding” a molecule if it is capable of specificallyreacting with the molecule to thereby bind the molecule to the antibody.

The term “antibody conjugate” refers to a binding protein, such as anantibody, chemically linked to a second chemical moiety, such as atherapeutic or cytotoxic agent. The term “agent” denotes a chemicalcompound, a mixture of chemical compounds, a biological macromolecule,or an extract made from biological materials. Particularly thetherapeutic or cytotoxic agents include, but are not limited to,pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogues or homologs thereof.

The term “antibody construct” as used herein refers to a polypeptidecomprising one or more the antigen binding portions of the inventionlinked to a linker polypeptide or an immunoglobulin constant domain.Linker polypeptides comprise two or more amino acid residues joined bypeptide bonds and are used to link one or more antigen binding portions.Such linker polypeptides are well known in the art (see e.g., Holliger,P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R.J., et al. (1994) Structure 2: 1121-1123). An immunoglobulin constantdomain refers to a heavy or light chain constant domain. Human IgG heavychain and light chain constant domain amino acid sequences are known inthe art and represented in Table 1.

TABLE 1 SEQUENCE OF HUMAN IgG HEAVY CHAIN CONSTANT DOMAIN ANDLIGHT CHAIN CONSTANT DOMAIN

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6: 93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31: 1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

The term “antigen-binding portion” or “antigen-binding fragment” of anantibody (or simply “antibody portion” or “antibody fragment”), as usedherein, refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., hRGM A). It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Such antibody embodiments mayalso be bispecific, dual specific, or multi-specific formats;specifically binding to two or more different antigens. Examples ofbinding fragments encompassed within the term “antigen-binding portion”of an antibody include (i) a Fab fragment, a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the VH andCH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341: 544-546, Winter et al., PCT publication WO 90/05144 A1herein incorporated by reference), which comprises a single variabledomain; and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123). Suchantibody binding portions are known in the art (Kontermann and DubelEds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.(ISBN 3-540-41354-5).

The term “antigenic determinant” or “epitope” includes any polypeptidedeterminant capable of specific binding to an immunoglobulin or T-cellreceptor. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments,may have specific three dimensional structural characteristics, and/orspecific charge characteristics. An epitope is a region of an antigenthat is bound by an antibody. In certain embodiments, an antibody issaid to specifically bind an antigen when it preferentially recognizesits target antigen in a complex mixture of proteins and/ormacromolecules.

The term “biological activity” refers to all inherent biologicalproperties of RGM A as defined herein.

The term “canonical residue” refers to a residue in a CDR or frameworkthat defines a particular canonical CDR structure as defined by Chothiaet al. (J. Mol. Biol. 196: 901-907 (1987); Chothia et al., J. Mol. Biol.227: 799 (1992), both are incorporated herein by reference). Accordingto Chothia et al., critical portions of the CDRs of many antibodies havenearly identical peptide backbone confirmations despite great diversityat the level of amino acid sequence. Each canonical structure specifiesprimarily a set of peptide backbone torsion angles for a contiguoussegment of amino acid residues forming a loop.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.The chimeric antibody can be produced through recombinant molecularbiological techniques, or may be physically conjugated together.

The term “CDR” refers to the complementarity determining region withinantibody variable sequences. There are three CDRs in each of thevariable regions of the heavy chain and the light chain, which aredesignated CDR1, CDR2 and CDR3, for each of the variable regions. Theterm “CDR set” as used herein refers to a group of three CDRs that occurin a single variable region capable of binding the antigen. The exactboundaries of these CDRs have been defined differently according todifferent systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia &Lesk, J. Mol. Biol. 196: 901-917 (1987) and Chothiaet al., Nature 342: 877-883 (1989)) found that certain sub-portionswithin Kabat CDRs adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3where the “L” and the “H” designates the light chain and the heavychains regions, respectively. These regions may be referred to asChothia CDRs, which have boundaries that overlap with Kabat CDRs. Otherboundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5): 732-45 (1996)). Still other CDR boundary definitions maynot strictly follow one of the above systems, but will nonethelessoverlap with the Kabat CDRs, although they may be shortened orlengthened in light of prediction or experimental findings thatparticular residues or groups of residues or even entire CDRs do notsignificantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, althoughparticular embodiments use Kabat or Chothia defined CDRs.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The terms “crystal”, and “crystallized” refer to an antibody, or antigenbinding portion thereof, that exists in the form of a crystal. Crystalsare one form of the solid state of matter, which is distinct from otherforms such as the amorphous solid state or the liquid crystalline state.Crystals are composed of regular, repeating, three-dimensional arrays ofatoms, ions, molecules (e.g., proteins such as antibodies), or molecularassemblies (e.g., antigen/antibody complexes). These three-dimensionalarrays are arranged according to specific mathematical relationshipsthat are well-understood in the field. The fundamental unit, or buildingblock, that is repeated in a crystal is called the asymmetric unit.Repetition of the asymmetric unit in an arrangement that conforms to agiven, well-defined crystallographic symmetry provides the “unit cell”of the crystal. Repetition of the unit cell by regular translations inall three dimensions provides the crystal. See Giege, R. and Ducruix, A.Barrett, Crystallization of Nucleic Acids and Proteins, a PracticalApproach, 2nd ea., pp. 20, 1-16, Oxford University Press, New York,N.Y., (1999).”

The terms “donor” and “donor antibody” refer to an antibody providingone or more CDRs. In a particular embodiment, the donor antibody is anantibody from a species different from the antibody from which theframework regions are obtained or derived. In the context of a humanizedantibody, the term “donor antibody” refers to a non-human antibodyproviding one or more CDRs.

The term “effective amount” refers to the amount of a therapy which issufficient to reduce or ameliorate the severity and/or duration of adisorder or one or more symptoms thereof, prevent the advancement of adisorder, cause regression of a disorder, prevent the recurrence,development, onset or progression of one or more symptoms associatedwith a disorder, detect a disorder, or enhance or improve theprophylactic or therapeutic effect(s) of another therapy (e.g.,prophylactic or therapeutic agent).

The term “framework” or “framework sequence” refers to the remainingsequences of a variable region minus the CDRs. Because the exactdefinition of a CDR sequence can be determined by different systems, themeaning of a framework sequence is subject to correspondingly differentinterpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain andCDR-H1, -H2, and -H3 of heavy chain) also divide the framework regionson the light chain and the heavy chain into four sub-regions (FR1, FR2,FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 andFR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Withoutspecifying the particular sub-regions as FR1, FR2, FR3 or FR4, aframework region, as referred by others, represents the combined FR'swithin the variable region of a single, naturally occurringimmunoglobulin chain. As used herein, a FR represents one of the foursub-regions, and FRs represents two or more of the four sub-regionsconstituting a framework region.

Human heavy chain and light chain acceptor sequences are known in theart. In one embodiment of the invention the human heavy chain and lightchain acceptor sequences are selected from the sequences described inTable 2 and Table 3. Different combinations for human frameworksequences FR1 to FR4 are stated in said tables.

TABLE 2 HUMAN HEAVY CHAIN ACCEPTOR SEQUENCES

TABLE 3 HUMAN LIGHT CHAIN ACCEPTOR SEQUENCES

The term “germline antibody gene” or “gene fragment” refers to animmunoglobulin sequence encoded by non-lymphoid cells that have notundergone the maturation process that leads to genetic rearrangement andmutation for expression of a particular immunoglobulin. (See, e.g.,Shapiro et al., Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchaloniset al., Adv Exp Med Biol. 484:13-30 (2001)). One of the advantagesprovided by various embodiments of the present invention stems from therecognition that germline antibody genes are more likely than matureantibody genes to conserve essential amino acid sequence structurescharacteristic of individuals in the species, hence less likely to berecognized as from a foreign source when used therapeutically in thatspecies.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like”, i.e., more similar tohuman germline variable sequences. One type of humanized antibody is aCDR-grafted antibody, in which human CDR sequences are introduced intonon-human VH and VL sequences to replace the corresponding nonhuman CDRsequences. Humanized antibody includes an antibody or a variant,derivative, analog or fragment thereof which immunospecifically binds toan antigen of interest and which comprises a framework (FR) regionhaving substantially the amino acid sequence of a human antibody and acomplementary determining region (CDR) having substantially the aminoacid sequence of a non-human antibody. As used herein, the term“substantially” in the context of a CDR refers to a CDR having an aminoacid sequence at least 50, 55, 60, 65, 70, 75 or 80%, particularly atleast 85%, at least 90%, at least 95%, at least 98% or at least 99%identical to the amino acid sequence of a non-human antibody CDR. Ahumanized antibody comprises substantially all of at least one, andtypically two, variable domains (Fab, Fab′, F(ab′)₂, FabC, Fv) in whichall or substantially all of the CDR regions correspond to those of anon-human immunoglobulin (i.e., donor antibody) and all or substantiallyall of the framework regions are those of a human immunoglobulinconsensus sequence. Particularly, a humanized antibody also comprises atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. In some embodiments, a humanizedantibody contains both the light chain as well as at least the variabledomain of a heavy chain. The antibody also may include the CH1, hinge,CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, ahumanized antibody only contains a humanized light chain. In someembodiments, a humanized antibody only contains a humanized heavy chain.In specific embodiments, a humanized antibody only contains a humanizedvariable domain of a light chain and/or humanized heavy chain.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgY, IgM, IgG, IgD, IgA and IgE, and anyisotype, including without limitation IgA1, IgA2, IgG1, IgG2, IgG3 andIgG4. The humanized antibody may comprise sequences from more than oneclass or isotype, and particular constant domains may be selected tooptimize desired effector functions using techniques well-known in theart.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In a particular embodiment,such mutations, however, will not be extensive. Usually, at least 50,55, 60, 65, 70, 75 or 80%, particularly at least 85%, more particularlyat least 90%, and most particularly at least 95% of the humanizedantibody residues will correspond to those of the parental FR and CDRsequences. As used herein, the term “consensus framework” refers to theframework region in the consensus immunoglobulin sequence. As usedherein, the term “consensus immuno-globulin sequence” refers to thesequence formed from the most frequently occurring amino acids (ornucleotides) in a family of related immunoglobulin sequences (See e.g.,Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany1987). In a family of immunoglobulins, each position in the consensussequence is occupied by the amino acid occurring most frequently at thatposition in the family. If two amino acids occur equally frequently,either can be included in the consensus sequence.

The term “human RGM A” (abbreviated herein as hRGM A) refers to aglycosylphosphatidyl-inositol (gpi)-anchored glycoprotein with 450 aminoacids, was first described as a neurite growth repellent or neuritegrowth inhibitor during development of topographic projections (Stahl etal. Neuron 5: 735-43, 1990; Mueller, in Molecular Basis of Axon Growthand Nerve Pattern Formation, Edited by H. Fujisawa, Japan ScientificSocieties Press, 215-229, 1997). The rgm gene family encompasses threedifferent genes, two of them, rgm a and b, are expressed in themammalian CNS, whereas the third member, rgm c, is expressed in theperiphery (Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci.361: 1513-29, 2006), where it plays an important role in ironmetabolism. Human RGM proteins have a sequence identity of 43%-50%; theamino acid homology of human and rat RGM A is 89%. Human RGM proteinsshare no significant sequence homology with any other known protein.They are proline-rich proteins containing an RGD region and havestructural homology to the Von-Willebrand-Factor domain and are cleavedat the N-terminal amino acid 168 by an unknown protease to yield thefunctionally active protein (Mueller et al., Philos. Trans. R. Soc.Lond. B Biol. Sci. 361: 1513-29, 2006).

In vitro, RGM A inhibits neurite outgrowth at picomolar concentrationsby binding to Neogenin, which has been identified as an RGM receptor(Rajagopalan et al. Nat Cell Biol.: 6(8), 756-62, 2004). Neogenin hadfirst been described as a netrin-binding protein (Keino-Masu et al.Cell, 87(2): 175-85, 1996), but its affinity for Netrin (K_(d) 2 nM) isan order of magnitude lower than that for RGM (K_(d) 0.2 nM)(Rajagopalan et al. Nat Cell Biol.: 6(8), 756-62, 2004). This is animportant finding because binding of Netrin-1 to Neogenin or to itsclosely related receptor DCC (deleted in colorectal cancer) has beenreported to stimulate rather than to inhibit neurite growth (Braisted etal. J. Neurosci. 20: 5792-801, 2000).

Besides binding of RGM A to Neogenin and inducing neurite growthinhibition, the binding of RGM A or B to the bone morphogenetic proteinsBMP-2 and BMP-4 could represent another obstacle to successfulneuroregeneration and functional recovery (Mueller et al., Philos.Trans. R. Soc. Lond. B Biol. Sci. 361: 1513-29, 2006). Both classes ofproteins (Neogenin and the BMPs) have been reported to transduce theneurite growth inhibitory signal of RGM A via two completely differentand independent signal transduction pathways. Usually, expression ofthese BMP proteins is relatively low in most regions of the adult CNS,but rapid increases in expression and accumulation of some BMPs (e.g.BMP-2, BMP-6, BMP-7) have been reported in response to injury and insult(Lai et al., Neuroreport 8: 2691-94, 1997; Martinez et al. Brain Res.894: 1-11, 2001; Hall and Miller, J. Neurosci. Res. 76: 1-8, 2004;Setoguchi et al., Exp. Neurol. 189: 33-44, 2004). In addition, in amodel of multiple sclerosis, the experimental autoimmuneencephalomyeltis (EAE) model, BMP-4, BMP-6 and BMP-7 were upregulated inmouse spinal cord (Ara et al., J. Neurosci. Res. 86: 125-35, 2008).BMP-2 has been reported to inhibit neurite growth by binding to cellsurface RGM A, BMP-receptors I and II and by directly activatingLIM-kinase (Matsuura et al. Biochem Biophys Res Commun., 360: 868-73,2007) and it is therefore expected that blocking the RGM A-BMP-2interaction will further increase functional recovery after CNS injury.

As mentioned above, spinally injured rats and humans with brain injury,show massive accumulations of cellular RGM at the injury site and thestaining pattern of RGM A in rats at the spinal lesion site is verysimilar to the pan RGM antibody staining in humans, suggesting that mostof the pan RGM staining in humans is related to RGM A localization butnot to RGM B localization (Schwab et al., Arch. Neurol. 62: 1561-8,2005a; Schwab et al. J. Neurosci. 21: 1569-76, 2005 b; Hata et al. J.Cell Biol. 173: 47-58, 2006). In healthy human brain, pan RGM staining(RGM A & B immunoreactivity) was detected on white matter fibers,oligodendrocytes, perikarya of few neurons, some vascular smooth muscleand few endothelial cells. No staining of astrocytes was observed. TheRGM staining pattern in adult healthy human brain is very similar to thestaining pattern observed in adult rat spinal cords (Schwab et al. Eur.J. Neurosci. 21: 1569-76, 2005 b; Hata et al. J. Cell Biol. 173: 47-58,2006).

Based on the accumulation of RGM A at lesion sites in brain and spinalcord injury and due to its cellular neurite growth inhibitory activity,it is expected that the protein will exert neurite growth inhibitoryactivity and its neutralization by antibodies, or antigen-bindingfragment thereof that bind to at least one epitope of the human RGM Amight result in improved regrowth of injured nerve fibers and in anenhancement of functional recovery in indications characterized by nervefiber injury and RGM accumulation.

The term “RGM A” also encompasses RGM A molecules isolated or obtainedfrom other species, as for example, rodents, like mice or rats;specifically, the rat derived molecule is designated herein as “rat RGMA”.

TABLE 4 LIST OF SEQUENCES OF RGM A RELATED MOLECULES Protein Sequenceidentifier Description hRGM A SEQ ID NO: 2 Human RGM A protein sequenceSEQ ID NO: 1 Human RGM A nucleotide sequence hRGM A SEQ ID NO: 4 HumanRGM A-fc protein sequence SEQ ID NO: 3 Human RGM A-fc nucleotidesequence hRGM A SEQ ID NO: 6 Human RGM A light chain - fc proteinsequence SEQ ID NO: 5 Human RGM A light chain - fc nucleotide sequencerat RGM A SEQ ID NO: 8 Rat RGM A protein sequence SEQ ID NO: 7 Rat RGM Anucleotide sequence

The term “inhibition of binding of RGM to one of its receptors”encompasses partial (as for example by about 20%, 40%, 60%, 80%, 85%,90%, 95% or more) or complete reduction of said receptor bindingactivity. Said “inhibition of binding” may be determined by any suitablemethod available in the art, particularly by any method as exemplifiedherein, as for example ELISA based binding assays.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hRGM A is substantially free of antibodies that specifically bindantigens other than hRGM A). An isolated antibody that specificallybinds hRGM A may, however, have cross-reactivity to other antigens, suchas RGM A molecules from other species. Moreover, an isolated antibodymay be substantially free of other cellular material and/or chemicals.

The term “isolated polynucleotide” shall mean a polynucleotide (e.g., ofgenomic, cDNA, or synthetic origin, or some combination thereof) that,by virtue of its origin, the “isolated polynucleotide”: is notassociated with all or a portion of a polynucleotide with which the“isolated polynucleotide” is found in nature; is operably linked to apolynucleotide that it is not linked to in nature; or does not occur innature as part of a larger sequence.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation is notassociated with naturally associated components that accompany it in itsnative state; is substantially free of other proteins from the samespecies; is expressed by a cell from a different species; or does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

The term “K_(d)” refers to the dissociation constant of a particularantibody-antigen interaction as is known in the art.

The term “key residues” refer to certain residues within the variableregion that have more impact on the binding specificity and/or affinityof an antibody, in particular a humanized antibody. A key residueincludes, but is not limited to, one or more of the following: a residuethat is adjacent to a CDR, a potential glycosylation site (can be eitherN- or O-glycosylation site), a rare residue, a residue capable ofinteracting with the antigen, a residue capable of interacting with aCDR, a canonical residue, a contact residue between heavy chain variableregion and light chain variable region, a residue within the Vernierzone, and a residue in the region that overlaps between the Chothiadefinition of a variable heavy chain CDR1 and the Kabat definition ofthe first heavy chain framework.

The term “k_(on)” refers to the on rate constant for association of anantibody to the antigen to form the antibody/antigen complex as is knownin the art.

The term “k_(off)” refers to the off rate constant for dissociation ofan antibody from the antibody/antigen complex as is known in the art.

The term “labelled binding protein” refers to a protein with a labelincorporated that provides for the identification of the bindingprotein. Particularly, the label is a detectable marker, e.g.,incorporation of a radiolabeled amino acid or attachment to apolypeptide of biotinyl moieties that can be detected by marked avidin(e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or colorimetric methods).Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm); fluorescent labels(e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, luciferase, alkaline phosphatase);chemiluminescent markers; biotinyl groups; predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags); and magnetic agents, such as gadoliniumchelates.

The term “local treatment” refers to any form of administration of thebinding protein of the invention or of a formulation containing the samedirectly at the intended region of the body of a mammal to be treatedwhere the desired neuroprotective or neuroregenerative action isintended to be induced.

The terms “modulate” and “regulate” are used interchangeably and referto a change or an alteration in the activity of a molecule of interest(e.g., the biological activity of hRGM A). Modulation may be an increaseor a decrease in the magnitude of a certain activity or function of themolecule of interest. Exemplary activities and functions of a moleculeinclude, but are not limited to, binding characteristics, enzymaticactivity, cell receptor activation, and signal transduction.

Correspondingly, the term “modulator” is a compound capable of changingor altering an activity or function of a molecule of interest (e.g., thebiological activity of hRGM A). For example, a modulator may cause anincrease or decrease in the magnitude of a certain activity or functionof a molecule compared to the magnitude of the activity or functionobserved in the absence of the modulator. The term “agonist”, as usedherein, refers to a modulator that, when contacted with a molecule ofinterest, causes an increase in the magnitude of a certain activity orfunction of the molecule compared to the magnitude of the activity orfunction observed in the absence of the agonist. Particular agonists ofinterest may include, but are not limited to, hRGM A polypeptides orpolypeptides, nucleic acids, carbohydrates, or any other molecules thatbind to hRGM A. The term “antagonist” as used herein, refer to amodulator that, when contacted with a molecule of interest causes adecrease in the magnitude of a certain activity or function of themolecule compared to the magnitude of the activity or function observedin the absence of the antagonist. Exemplary antagonists include, but arenot limited to, proteins, peptides, antibodies, peptibodies,carbohydrates or small organic molecules. Peptibodies are described,e.g., in WO01/83525.

Particular antagonists of interest include those that block or modulatethe biological or immunological activity of hRGM A. Antagonists of hRGMA may include, but are not limited to, proteins, nucleic acids,carbohydrates, or any other molecules, which bind to hRGM A, likemonoclonal antibodies that interact with the RGM A molecule. It shouldbe noted that the interaction with RGM A may result in binding andneutralization of other ligands/cell membrane components, and may beuseful for additive or synergistic functioning against multiplediseases.

The term “monoclonal antibody” refers to a preparation of antibodymolecules, which share a common heavy chain and common light chain aminoacid sequence, in contrast with “polyclonal” antibody preparations thatcontain a mixture of different antibodies. Monoclonal antibodies can begenerated by several novel technologies like phage, bacteria, yeast orribosomal display, as well as classical methods exemplified byhybridoma-derived antibodies (e.g., an antibody secreted by a hybridomaprepared by hybridoma technology, such as the standard Kohler andMilstein hybridoma methodology ((1975) Nature 256: 495-497).

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2) or subclass.

The term “neuroprotective” or “neuroprotection” refers to a treatmentwhich prevents neuronal cells, in particular cells comprising cells ofthe RNFL from damage or degeneration, (analyzable by observingpreservation of RGC (Retinal Ganglion Cell) axons in the retina fromdegeneration; or inhibition of “abnormal” RNFL degeneration measurablevia RNFL thickness losses as defined above.

The term “neuroregenerative” or “neuroregeneration” refers to atreatment according to the invention which results in a repair orre-growth of neuronal cells, in particular cells comprising cells of adamaged RNFL, (analyzable by, for example, Optic Coherence Tomography(OCT) of retina, diffusion tensor imaging of optic nerves; retinalneuron sprouting;

The term “neutralizing” refers to neutralization of biological activityof a target protein when a binding protein specifically binds the targetprotein. Neutralizing may be the result of different ways of binding ofsaid binding protein to the target. For example, neutralizing may becaused by binding of the binding protein in a region of the target whichdoes not affect receptor binding to the target molecule. Alternativelybinding of a binding protein may result in a blockade of the receptorbinding to the target, which blockade finally neutralizes the targetprotein activity. A neutralizing binding protein is a neutralizingantibody whose binding to hRGM A results in neutralization of abiological activity of hRGM A. Particularly the neutralizing bindingprotein binds hRGM A and decreases a biologically activity of hRGM A byat least about 20%, 40%, 60%, 80%, 85% or more. Neutralization of abiological activity of hRGM A by a neutralizing binding protein can beassessed by measuring one or more indicators of hRGM A biologicalactivity well known in the art. For example neutralization of hRGM Areverses the inhibition in a Ntera neuronal outgrowth assay (see Example3, below). The Ntera neurite growth assay addresses inhibition ofneurite outgrowth. In the absence of an inhibitory RGM A protein orfragment and in the presence of the outgrowth-stimulating substratelaminin, neuronal NTera aggregates show an extensive and dense networkof outgrowing neurites. RGM A or RGM A fragments inhibit neuriteoutgrowth, resulting in reduced length and numbers of neurites.Function-blocking RGM A antagonists or MABs like mAb 5F9 neutralized theneurite outgrowth inhibitory activity of the potent fc-conjugated hRGM Alight chain fragment (amino acids 47-168) of the human RGM A protein inneurite growth assays with aggregates of differentiated human NTeraneurons, resulting in a strong increase in neurite length and numbers.

The term “neutralizing monoclonal antibody” refers to a preparation ofantibody molecules, which upon binding to the specific antigen are ableto compete and inhibit the binding of the natural ligand for saidantigen. In a particular embodiment of the present application, theneutralizing antibodies of the present invention are capable ofcompeting with RGM A for binding to Neogenin and/or to BMP-2 and/orBMP-4, and to prevent RGM A biological activity or function. Inparticular, the neutralizing antibodies of the present invention arecapable of binding with RGM A and to prevent binding to Neogenin and/orto BMP-2 and/or BMP-4, and to prevent RGM A biological activity orfunction. The term “activity” includes activities such as the bindingspecificity/affinity of an antibody for an antigen, for example, ananti-hRGM A antibody that binds to an RGM A antigen and/or theneutralizing potency of an antibody, for example, an anti-hRGM Aantibody whose binding to hRGM A inhibits the biological activity ofhRGM A, e.g. as determined in a hRGM A-Neogenin binding assay, hRGMA-BMP-2 binding assay or hRGM A-BMP-4 binding assay as described belowin the experimental section.

The biologic activity of RGM A can be described as regulating cellularmigration. A special example of cellular migration is neurite growth,which is impeded or inhibited by RGM A proteins. In addition RGMproteins have been shown to modulate activity of BMP-proteins. Hereinpublished examples describe a synergizing, potentiating activity of RGMproteins on the BMP-pathway on one side and an inhibitory activity ofRGM proteins on the BMP-pathway, which is important for regulation ofiron metabolism, bone and cartilage regeneration and in the CNS forremyelination and regeneration.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” as used hereinrefers to polynucleotide sequences, which are necessary to effect theexpression and processing of coding sequences to which they are ligated.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

The term “polynucleotide” means a polymeric form of two or morenucleotides, either ribonucleotides or deoxynucleotides or a modifiedform of either type of nucleotide. The term includes single and doublestranded forms of DNA but particularly is double-stranded DNA.

The term “polypeptide” refers to any polymeric chain of amino acids. Theterms “peptide” and “protein” are used interchangeably with the termpolypeptide and also refer to a polymeric chain of amino acids. The term“polypeptide” encompasses native or artificial proteins, proteinfragments and polypeptide analogs of a protein sequence. A polypeptidemay be monomeric or polymeric.

The term “recombinant host cell” (or simply “host cell”) is intended torefer to a cell into which exogenous DNA has been introduced. It shouldbe understood that such terms are intended to refer not only to theparticular subject cell, but, to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein. Particularly host cells includeprokaryotic and eukaryotic cells selected from any of the Kingdoms oflife. Particular eukaryotic cells include protist, fungal, plant andanimal cells. Most particularly host cells include but are not limitedto the prokaryotic cell line E. coli; mammalian cell lines CHO, HEK 293and COS; the insect cell line Sf9; and the fungal cell Saccharomycescerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (Hoogenboom H. R., (1997) TIB Tech. 15: 62-70;Azzazy H., and Highsmith W. E., (2002) Clin. Biochem. 35: 425-445;Gavilondo J. V., and Larrick J. W. (2002) BioTechniques 29: 128-145;Hoogenboom H., and Chames P. (2000) Immunology Today 21: 371-378),antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992)Nucl. Acids Res. 20: 6287-6295; Kellermann S-A., and Green L. L. (2002)Current Opinion in Biotechnology 13: 593-597; Little M. et al (2000)Immunology Today 21: 364-370) or antibodies prepared, expressed, createdor isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

The term “recovering” refers to the process of rendering a chemicalspecies such as a polypeptide substantially free of naturally associatedcomponents by isolation, e.g., using protein purification techniqueswell known in the art.

The term “RNFL” is the innermost layer of the retina and is principallycomposed of axons from ganglion cell neurons that compose the opticnerve. The rentinal layer structure comprises said innermost RNFLfollowed by inner plexiform layer (IPL), the outer plexiform layer(OPL), the photoreceptor layer (PRL) and the retinal pigment epithelium(RPE). In healthy individuals, the RNFL is only about 110-120 μm thickby the age of 15 years and most normal individuals will lose about0.017% per year in retinal thickness which equates to about 10-20 μmover 60 years (changes in the thickness of RNFL may, for example, bedetermined by the OCT method as described by Frohman et al, above). Saidloss of thickness may also be designated “normal” RNFL degeneration.

A degeneration or loss of thickness above or greater than said “normal”degeneration may also be designated as “abnormal” or “disease-related”RNFL degeneration. In particular such abnormal losses of thickness maybe above 0.017%, as for example above 0.02%, or above 0.1%, or above 1%,or above 5%, or above 10% or above 20% per year, as for example 1 to 10%or 2 to 5% per months.

The term “RNFL degeneration” encompasses both normal and, in particular,abnormal, disease-related thickness losses of the RNFL.

The term “sample” is used in its broadest sense. A “biological sample”,as used herein, includes, but is not limited to, any quantity of asubstance from a living thing or formerly living thing. Such livingthings include, but are not limited to, humans, mice, rats, monkeys,dogs, rabbits and other animals. Such substances include, but are notlimited to, blood, serum, urine, synovial fluid, cells, organs, tissues,bone marrow, lymph nodes and spleen.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, mean that the interaction is dependentupon the presence of a particular structure (e.g., an “antigenicdeterminant” or “epitope” as defined below) on the chemical species; forexample, an antibody recognizes and binds to a specific proteinstructure rather than to proteins generally. If an antibody is specificfor epitope “A”, the presence of a molecule containing epitope A (orfree, unlabeled A), in a reaction containing labeled “A” and theantibody, will reduce the amount of labeled A bound to the antibody.

The term “surface plasmon resonance” refers to an optical phenomenonthat allows for the analysis of real-time biospecific interactions bydetection of alterations in protein concentrations within a biosensormatrix, for example using the BIAcore system (Pharmacia Biosensor AB,Uppsala, Sweden and Piscataway, N.J.). For further descriptions, seeJönsson, U., et al. (1993) Ann. Biol. Clin. 51: 19-26; Jönsson, U., etal. (1991) Biotechniques 11: 620-627; Johnsson, B., et al. (1995) J.Mol. Recognit. 8: 125-131; and Johnnson, B., et al. (1991) Anal.Biochem. 198: 268-277.

The term “systemic treatment” refers to any form of administration ofthe binding protein of the invention or of a formulation containing thesame to the body of a mammal to be treated so that the binding proteinreaches the intended place of neuroprotective or neuroregenerativeaction via the bloodstream. For example, systemic administrationencompasses infusion or injection of the binding protein of theinvention or a formulation containing the same.

The term “transformation” refers to any process by which exogenous DNAenters a host cell. Transformation may occur under natural or artificialconditions using various methods well known in the art. Transformationmay rely on any known method for the insertion of foreign nucleic acidsequences into a prokaryotic or eukaryotic host cell. The method isselected based on the host cell being transformed and may include, butis not limited to, viral infection, electroporation, lipofection, andparticle bombardment. Such “transformed” cells include stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome. They also include cells which transiently express theinserted DNA or RNA for limited periods of time.

The term “transgenic organism” refers to an organism having cells thatcontain a transgene, wherein the transgene introduced into the organism(or an ancestor of the organism) expresses a polypeptide not naturallyexpressed in the organism. A “transgene” is a DNA construct, which isstably and operably integrated into the genome of a cell from which atransgenic organism develops, directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicorganism.

The term “treatment of RNFL degeneration” refers to both the therapeutic(i.e. acute) and the prophylactic, treatment of RNFL degeneration. Saidtreatment may be “neuroregenerative” or “neuroprotective”; saidtreatment may be either in the form of a “local” or a “systemic”treatment.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “expressionvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The term “vernier zone” refers to a subset of framework residues thatmay adjust CDR structure and fine-tune the fit to antigen as describedby Foote and Winter (1992, J. Mol. Biol. 224: 487-499, which isincorporated herein by reference). Vernier zone residues form a layerunderlying the CDRs and may impact on the structure of CDRs and theaffinity of the antibody.

2. Use of RGM a Antibodies

The neutralizing monoclonal antibody against RGM A, selectively inhibitsbinding of RGM A to its receptor Neogenin and to bone morphogeneticproteins 2 and 4 (BMP-2, BMP-4). The neutralizing monoclonal antibodiesstimulate regrowth of injured or damaged nerve fibers and formation offunctional synapses of regenerating nerve fibers, and to inducelong-distance regeneration in an in vivo rat model of optic nerve injuryand it also enhances remyelination of lesioned and regenerating nervefibers (see PCT/EP2009/001437).

Surprisingly, it was observed that the antibodies against RGM A areassociated with an additional not yet recognized direct therapeuticeffect on the RNFL. In particular, patients suffering from an oculardisease associated with RNFL degeneration or bearing the risk of RNFLdegeneration may be treated with an antibody molecule as defined herein.The present invention is also based on the surprising finding thatsystemically administered antibodies of the present invention arelocalized in the retina and exert their beneficial effect directly atthe diseased region of the eye.

Thus, for the first time, a method of directed treatment of a specificgroup of patients aiming at the protection against RNFL degeneration oraiming at regeneration of already degenerated RNFL using antibodiescapable of binding RGM A is provided herein.

Another embodiment relates to neuroprotective treatment of RNFLdegeneration as observed in a multiplicity of disease conditions usingantibodies capable of binding RGM A.

Another embodiment relates to the use of a monoclonal antibody thatblocks RGM A and prevents the interaction between RGM A and its receptorand/or binding proteins, i.e. Neogenin and BMP-2, BMP-4, for thetreatment of RNFL degeneration.

Another embodiment of the invention relates to binding protein for humanRGM A for use in the treatment of RNFL degeneration.

In another embodiment, said treatment is an therapeutic or prophylactic,neuroregenerative or neuroprotective, local or systemic treatment.

In another embodiment, as a result of said treatment

-   -   a. retinal neuron sprouting is observed; and/or    -   b. RGC (Retinal Ganglion Cell) axons in the retina are preserved        from degeneration.

According to one aspect, the present invention provides a bindingprotein that dissociates from human RGM A (hRGM A) with a K_(D) of1×10⁻⁷ M or less and a k_(off) rate constant of 1×10⁻² s⁻¹ or less, bothdetermined by surface plasmon resonance for use in the treatment of RNFLdegeneration.

According to another aspect, the invention relates to a binding protein,as for example a binding protein showing the above kinetic features,that binds to human RGM A and neutralizes the neurite outgrowthinhibitory activity of human RGM A as determined in a standard in vitroassay, as for example the Ntera neuronal outgrowth assay as exemplifiedin Example 3, below for use in the treatment of RNFL degeneration.

Another embodiment also relates to a binding protein for a use asdefined above, having at least one of the following additionalfunctional characteristics:

-   -   binding to rat RGM A,    -   binding to human RGM C, and    -   binding to rat RGM C.

In another embodiment, the binding protein as defined herein modulatesthe ability of RGM to bind to at least one of its receptors. Suchbinding protein binds to a receptor binding domain of human RGM A. ForRGM A a N- and a C-terminal receptor binding domains have beenidentified. Particular embodiments of the binding proteins of theinvention bind to the N-terminal receptor binding domain of RGM A, asillustrated by the inhibition of binding between an N-terminal hRGM Afragment, as for example 47-168 and receptor molecules, like Neogeninand BMP-4. Said N-terminal hRGM A fragment may have a total length ofabout 30 to about 150 or about 30 to about 122 amino acid residues. As anon-limiting example Fragment 0 (corresponding to the N-terminalresidues 47-168) of hRGM A as described herein or any shorter receptorbinding fragment may be mentioned.

In another embodiment said binding protein modulates or inhibits, atleast one of the following interactions:

-   -   binding of human RGM A to human BMP-4.    -   binding of hRGM A to human Neogenin,    -   binding of hRGM C to human Neogenin,    -   binding of human RGM A to human BMP-2.

According to a particular embodiment, the binding protein as hereindefined is a humanized antibody.

The binding protein as described above may have an antigen bindingdomain, said binding protein capable of binding an epitope of an RGMmolecule, said antigen binding domain comprising at least one CDRcomprising an amino acid sequence selected from the group consisting of

GTTPDY, (SEQ ID NO: 59) FQATHDPLT, (SEQ ID NO: 62) ARRNEYYGSSFFDY,(SEQ ID NO: 65) LQGYIPPRT, (SEQ ID NO: 68)andmodified CDR amino acid sequences having a sequence identity of at least50% to one of said sequences. In another embodiment the presentinvention relates to a binding protein, comprising an antigen bindingdomain, said binding protein being capable of binding an epitope of anRGM molecule, said antigen binding domain comprising at least one CDRcomprising an amino acid sequence selected from the group consisting of:

GTTPDY, (SEQ ID NO: 59) FQATHDPLT, (SEQ ID NO: 62) ARRNEYYGSSFFDY,(SEQ ID NO: 65) LQGYIPPRT, (SEQ ID NO: 68)andmodified CDR amino acid sequences having a sequence identity of at least50%, as for example at least 55, 60, 65, 70, 75, 80, 85, 90, 95%identity to one of said sequences.

For example, said binding protein may comprise two of said CDRs, as forexample SEQ ID NO:59 and 62; or SEQ ID NO:65 and 68; wherein at leastone of said CDRs may be modified, having a sequence identity of at least50%, as for example at least 55, 60, 65, 70, 75, 80, 85, 90, 95%identity to one of said sequences.

Said binding protein may further comprise at least one CDR comprise anamino acid sequence selected from the group consisting of SEQ ID NO:57,58, 60, 61, 63, 64, 66, 67 and modified CDR amino acid sequences havinga sequence identity of at least 50%, as for example at least 55, 60, 65,70, 75, 80, 85, 90, 95% identity, to one of said sequences.

In another embodiment, said at least one CDR comprises an amino acidsequence selected from the group consisting of:

SEQ ID NO: 57 Residues 31-35 of SEQ ID NO.: 34 SEQ ID NO: 58 Residues50-66 of SEQ ID NO.: 34 SEQ ID NO: 59 Residues 99-104 of SEQ ID NO.: 34SEQ ID NO: 60 Residues 24-39 of SEQ ID NO.: 10 SEQ ID NO: 61 Residues55-61 of SEQ ID NO.: 10 SEQ ID NO: 62 Residues 94-102 of SEQ ID NO.: 10SEQ ID NO: 63 Residues 31-35 of SEQ ID NO.: 55 SEQ ID NO: 64 Residues50-66 of SEQ ID NO.: 55 SEQ ID NO: 65 Residues 97-110 of SEQ ID NO.: 55SEQ ID NO: 66 Residues 24-34 of SEQ ID NO.: 56 SEQ ID NO: 67 Residues50-56 of SEQ ID NO.: 56 SEQ ID NO: 68 Residues 89-97 of SEQ ID NO.: 56

In another embodiment, said binding protein comprises at least 3 CDRswhich are selected from a variable domain CDR set consisting of:

VH 5F9 set VH 5F9 CDR-H1 Residues 31-35 of SEQ ID NO.: 34 SEQ ID NO: 57VH 5F9 CDR-H2 Residues 50-66 of SEQ ID NO.: 34 SEQ ID NO: 58 VH 5F9CDR-H3 Residues 99-104 of SEQ ID NO: 59 SEQ ID NO.: 34 VL 5F9 set VL 5F9CDR-L1 Residues 24-39 of SEQ ID NO.: 10 SEQ ID NO: 60 VL 5F9 CDR-L2Residues 55-61 of SEQ ID NO.: 10 SEQ ID NO: 61 VL 5F9 CDR-L3 Residues94-102 of SEQ ID NO: 62 SEQ ID NO.: 10 VH 8D1 set VH 8D1 CDR-H1 Residues31-35 of SEQ ID NO.: 55 SEQ ID NO: 63 VH 8D1 CDR-H2 Residues 50-66 ofSEQ ID NO.: 55 SEQ ID NO: 64 VH 8D1 CDR-H3 Residues 97-110 of SEQ ID NO:65 SEQ ID NO.: 55 VL 8D1 set VL 8D1 CDR-L1 Residues 24-34 of SEQ ID NO.:56 SEQ ID NO: 66 VL 8D1 CDR-L2 Residues 50-56 of SEQ ID NO.: 56 SEQ IDNO: 67 VL 8D1 CDR-L3 Residues 89-97 of SEQ ID NO: 68 SEQ ID NO.: 57or a variable domain set wherein at least one of said 3 CDRs is amodified CDR amino acid sequence having a sequence identity of at least50%, as for example at least 55, 60, 65, 70, 75, 80, 85, 90, 95%identity, to the parent sequence.

Each of said above mentioned modifications may be generated by single ormultiple amino acid addition, deletion, or, in particular, substitution,or combinations thereof.

In another embodiment, the binding protein comprises at least twovariable domain CDR sets.

Said at least two variable domain CDR sets are selected from a groupconsisting of:

-   -   VH 5F9 set& VL 5F9 set; and    -   VH 8D1 set & VL 8D1 set

The binding protein as used according to another embodiment of theinvention further comprises a human acceptor framework.

Said human acceptor framework may comprise at least one amino acidsequence selected from the group consisting of SEQ ID NO: 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and 33.

The binding protein of the invention may comprise at least one set offramework sequences selected from the group consisting of the sets:

(1) VH3-48 set (SEQ ID NO: 15, 16 and 17)

VH3-33 set (SEQ ID NO: 21, 22 and 23)

VH3-23 set (SEQ ID NO: 24, 25 and 26)

-   -   each of which sets being combined with a further framework        sequence, selected from    -   JH3 (SEQ ID NO:18),    -   JH4 (SEQ ID NO:19),    -   JH6 (SEQ ID NO:20);

or

(2) selected from the group consisting of the sets

A18 set: (SEQ ID NO:27, 28 and 29)

A17 set: (SEQ ID NO:31, 32 and 33)

-   -   each of which sets being combined with a further framework        sequence, selected from JK2 (SEQ ID NO:2)

The binding protein as defined above comprising at least one CDR-graftedheavy chain variable domain selected from SEQ ID NO:35, 36, 37, 38, 39,40, 41, 42, and 43; and/or at least one CDR-grafted light chain variabledomain selected from SEQ ID NO:44, 45, and 46.

The binding protein as used according to another embodiment of theinvention comprises a combination of two variable domains, wherein saidtwo variable domains have amino acid sequences selected from:

SEQ ID NOs: 35 & 44; 36 & 44; 37 & 44; 38 & 44; 39 & 44; 40 & 44; 41 &44; 42 & 44; 43 & 44;

SEQ ID NOs: 35 & 45; 36 & 45; 37 & 45; 38 & 45; 39 & 45; 40 & 45; 41 &45; 42 & 45, 43 & 45

SEQ ID NOs: 35 & 46; 36 & 46; 37 & 46; 38 & 46; 39 & 46; 40 & 46; 41 &46; 42 & 46; 43 & 46;

In another embodiment of the invention, said human acceptor framework ofthe binding protein as used according to the invention comprises atleast one framework region amino acid substitution at a key residue,said key residue selected from the group consisting of:

-   -   a residue adjacent to a CDR;    -   a glycosylation site residue;    -   a rare residue;    -   a residue capable of interacting with a RGM epitope;    -   a residue capable of interacting with a CDR;    -   a canonical residue;    -   a contact residue between heavy chain variable region and light        chain variable region;    -   a residue within a Vernier zone;    -   an N-terminal residue capable of paraglutamate formation and    -   a residue in a region that overlaps between a Chothia-defined        variable heavy chain CDR1 and a Kabat-defined first heavy chain        framework.

Said key residues are selected from the group consisting

-   -   (heavy chain sequence position): 1, 5, 37, 48, 49, 88, 98    -   (light chain sequence position): 2, 4, 41, 51

In a particular embodiment, the binding protein as used according toanother embodiment of the invention is or comprises a consensus humanvariable domain.

According to another embodiment of the binding protein as used accordingto another embodiment of the invention, said human acceptor frameworkcomprises at least one framework region amino acid substitution, whereinthe amino acid sequence of the framework is at least 65%, as for exampleat least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99%, identical to thesequence of said human acceptor framework and comprises at least 70amino acid residues, as for example at least 75, 80, or 85 residues,identical to said human acceptor framework.

According to particular embodiment, the binding protein as usedaccording to another embodiment of the invention comprises at least oneframework mutated variable domain having an amino acid sequence selectedfrom the group consisting of:

SEQ ID NO:47, 48, 49, 50; (VH domain), and/or

selected from the group consisting of:

SEQ ID NO: 51, 52, 53, and 54 (VL domain)

In particular, said binding protein comprises two optionally frameworkmutated variable domains, wherein said two variable domains have aminoacid sequences selected from the groups consisting of:

SEQ ID NOs:47 & 44; 47 & 45; 47 & 46; 47 & 51; 47 & 52; 47 & 53; 47 &54;

SEQ ID NOs:48 & 44; 48 & 45; 48 & 46; 48 & 51; 48 & 52; 48 & 53; 48 &54;

SEQ ID NOs:49 & 44; 49 & 45; 49 & 46; 49 & 51; 49 & 52; 49 & 53; 49 &54;

SEQ ID NOs:50 & 44; 50 & 45; 50 & 46; 50 & 51; 50 & 52; 50 & 53; 50 &54;

The binding proteins as used according to another embodiment of theinvention as described herein are capable of binding at least onetarget, selected from RGM molecules. They are capable of binding tohuman RGM A, and optionally at least one further RGM molecule of humanorigin or originating from cynomolgus monkeys, rat, chick, frog, andfish. For example they may additionally bind to rat RGM A, human RGM C,and/or rat RGM C.

The binding protein as used according to another embodiment of theinvention is capable of modulating, in particular, capable ofneutralizing or inhibiting a biological function of a target, selectedfrom RGM molecules as defined above.

In particular, the binding protein as used according to anotherembodiment of the invention modulates, in particular inhibits, theability of RGM to bind to at least one of its receptors, as for exampleNeogenin, and BMP, like BMP-2 and BMP-4. For example said bindingprotein modulates, in particular diminishes and particularly inhibits atleast one of the following interactions:

-   -   binding of human RGM A to human BMP-4.    -   binding of hRGM A to human Neogenin,    -   binding of hRGM C to human Neogenin,    -   binding of human RGM A to human BMP-2.

Binding proteins with different combinations of functional features, andconsequently showing different functional profiles, as disclosed herein,are also within the scope of the invention. Non-limiting examples ofsuch profiles are listed below:

Profiles Feature 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2122 Binding + + + + + + + + + + + + + + + + + + + + + + to human RGM ABinding + − + − + + − + − + − + − + − + − + − + + to rat RGM A Binding +− − − − + − − + + − − + + − − + + − − + + to human RGM C Binding + − − −− − − − − − + + + + − − − − + + + + to rat RGM C Inhibition + −− + + + + + + + + + + + + + + + + + + + of binding of hRGM A to humanNeogenin Inhibition + − − − − + − − − − − − − − + + + + + + + + ofbinding of hRGM C to human Neogenin Inhibition + + + + + + − − − − − − −− − − − − − − − − of binding of human RGM A to human BMP-2Inhibition + + + + + + − − − − − − − − − − − − − − − − of binding ofhuman RGM A to human BMP-4

For example, profile 1 is met by antibody 5F9 as provided by the presentinvention and its derivatives described herein.

For example, profile 2 is met by antibody 8D1 as provided by the presentinvention and its derivatives as disclosed herein.

A binding protein as used according to another embodiment of theinvention is capable of inhibiting at least one biological activity ofRGM, in particular RGM A, wherein said RGM A is selected from human,cynomolgus monkeys, rat, chick, frog, and fish.

The binding protein as used according to another embodiment of theinvention has one or more of the following kinetic features:

-   -   (a) an on rate constant (k_(on)) to said target selected from        the group consisting of: at least about 10²M⁻¹ s⁻¹; at least        about 10³M⁻¹ s⁻¹; at least about 10⁴M⁻¹ s⁻¹; at least about        10⁵M⁻¹ s⁻¹; at least about 10⁶M⁻¹ s⁻¹, and at least about 10⁷M⁻¹        s⁻¹, as measured by surface plasmon resonance;    -   (b) an off rate constant (k_(off)) to said target selected from        the group consisting of: at most about 10⁻² s⁻¹, at most about        10⁻³ s⁻¹; at most about 10⁻⁴ s⁻¹; at most about 10⁻⁵ s⁻¹; and at        most about 10⁻⁶ s⁻¹, as measured by surface plasmon resonance;        or    -   (c) a dissociation constant (K_(D)) to said target selected from        the group consisting of: at most about 10⁻⁷ M; at most about        10⁻⁸ M; at most about 10⁻⁹ M; at most about 10⁻¹⁰ M; at most        about 10⁻¹¹ M; at most about 10⁻¹² M; and at most 10⁻¹³M.

According to a further aspect, the present invention makes use of anantibody construct comprising a binding protein described above, saidantibody construct further comprising a linker polypeptide or animmunoglobulin constant domain.

Said antibody construct or binding protein may be selected from thegroup consisting of: an immunoglobulin molecule, a monoclonal antibody,a chimeric antibody, a CDR-grafted antibody, a humanized antibody, aFab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, a singledomain antibody, a diabody, a multispecific antibody, a dual specificantibody, a dual variable domain immunoglobulin, and a bispecificantibody.

In an antibody construct as used according to another embodiment of theinvention said binding protein comprises a heavy chain immunoglobulinconstant domain selected from the group consisting of;

a human IgM constant domain,

a human IgG1 constant domain,

a human IgG2 constant domain,

a human IgG3 constant domain,

a human IgG4 constant domain,

a human IgE constant domain,

a human IgD constant domain,

a human IgA1 constant domain

a human IgA2 constant domain

a human IgY constant domain and

corresponding mutated constant domains

An antibody construct as used according to another embodiment of theinvention comprises an immunoglobulin constant domain having an aminoacid sequence selected from the group consisting of: SEQ ID NO: 11, 12,13 and 14

According to another aspect, the present invention makes use of anantibody conjugate comprising an antibody construct described herein,said antibody conjugate further comprising an agent selected from thegroup consisting of; an immunoadhesion molecule, an imaging agent, atherapeutic agent, and a cytotoxic agent, each of which agent beingconjugated, of example covalently bound to said binding protein.

For example, said agent is an imaging agent selected from the groupconsisting of a radiolabel, an enzyme, a fluorescent label, aluminescent label, a bioluminescent label, a magnetic label, and biotin.In particular, said imaging agent is a radiolabel selected from thegroup consisting of: ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu,¹⁶⁶Ho and ¹⁵³Sm.

For example, said agent is a therapeutic or cytotoxic agent selectedfrom the group consisting of; an anti-metabolite, an alkylating agent,an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, ananti-mitotic agent, an anthracycline, toxin, and an apoptotic agent.

According to another embodiment, said binding protein as used accordingto another embodiment of the invention possesses a human glycosylationpattern.

Furthermore, the binding proteins, antibody constructs and antibodyconjugates as used according to another embodiment of the invention mayexist as a crystal (in crystalline form), particularly retainingbiological activity. Said crystal is a carrier-free pharmaceuticalcontrolled release crystal. In view of said crystalline form the bindingprotein, antibody construct or antibody conjugate may have a greaterhalf life in vivo than the corresponding soluble counterpart.

In another aspect the present invention provides an isolated nucleicacid encoding a binding protein amino acid sequence, antibody constructamino acid sequence, and antibody conjugate amino acid sequence asdescribed herein.

Another embodiment of the invention also relates to a vector comprisingan isolated nucleic acid as described herein. In particular, the vectoris selected from the group consisting of pcDNA, pTT, pTT3, pEFBOS, pBV,pJV, and pBJ.

Another embodiment of the invention also relates to a host cellcomprising such a vector. In particular, said host cell is a prokaryoticcell, as for example E. coli; or is a eukaryotic cell, and may beselected from the group consisting of protist cell, animal cell, plantcell and fungal cell. In particular, said eukaryotic cell is an animalcell selected from the group consisting of a mammalian cell, an aviancell, and an insect cell. Said host cell is selected from HEK cells, CHOcells, COS cells and yeast cells. The yeast cell may be Saccharomycescerevisiae and said insect cell may be a Sf9 cell.

Another embodiment of the invention also provides a method of producinga protein capable of binding RGM, comprising culturing a host cell asdefined herein in culture medium under conditions sufficient to producea binding protein capable of binding RGM.

Another embodiment of the invention also relates to a protein producedaccording to said method.

Another embodiment provides a composition for the release of a bindingprotein said composition comprising

-   -   (a) a formulation, wherein said formulation comprises a        crystallized product protein as defined herein, and an        ingredient; and    -   (b) at least one polymeric carrier.

Said polymeric carrier may be a polymer selected from one or more of thegroup consisting of: poly (acrylic acid), poly (cyanoacrylates), poly(amino acids), poly (anhydrides), poly (depsipeptide), poly (esters),poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly(b-hydroxybutryate), poly (caprolactone), poly (dioxanone); poly(ethylene glycol), poly ((hydroxypropyl)methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers,pluronic polyols, albumin, alginate, cellulose and cellulosederivatives, collagen, fibrin, gelatin, hyaluronic acid,oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends andcopolymers thereof.

Said ingredient may be selected from the group consisting of albumin,sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin,methoxypolyethylene glycol and polyethylene glycol.

According to another aspect the present invention provides a method fortreating a mammal comprising the step of administering to the mammal aneffective amount of the composition as defined herein.

According to another aspect the present invention provides apharmaceutical composition comprising the product (in particular, abinding protein, construct or conjugate as scribed herein above), and apharmaceutically acceptable carrier.

Said pharmaceutically acceptable carrier may function as adjuvant usefulto increase the absorption, or dispersion of said binding protein.

For example said adjuvant is hyaluronidase.

According to another embodiment said pharmaceutical further comprises atleast one additional therapeutic agent for treating a disorder in whichRGM activity is detrimental. For example said agent is selected from thegroup consisting of: therapeutic agent, imaging agent, cytotoxic agent,angiogenesis inhibitors; kinase inhibitors; co-stimulation moleculeblockers; adhesion molecule blockers; anti-cytokine antibody orfunctional fragment thereof; methotrexate; cyclosporin; rapamycin;FK506; detectable label or reporter; a TNF antagonist; an antirheumatic;a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, aneuromuscular blocker, an antimicrobial, an antipsoriatic, acorticosteriod, an anabolic steroid, an erythropoietin, an immunization,an immunoglobulin, an immunosuppressive, a growth hormone, a hormonereplacement drug, a radiopharmaceutical, an antidepressant, anantipsychotic, a stimulant, an asthma medication, a beta agonist, aninhaled steroid, an epinephrine or analogue, a cytokine, and a cytokineantagonist.

The present invention also relates to a method for treating a subjectfor a disorder associated with RNFL degeneration comprising the step ofadministering alone or in combination with other therapeutic agents aproduct of the invention (in particular, a binding protein, construct orconjugate as described herein above).

Said disorder particularly comprises diseases selected from the groupcomprising diabetic retinopathy, ischemic optic neuropathy, X-chromosomelinked retinoschisis, drug-induced optic neuropathy, retinal dystrophy,age-related macula degeneration, eye diseases characterized by opticnerve head drusen, eye disease characterized by genetic determinants ofphotoreceptor degeneration, autosomal recessive cone-rod dystrophy, andmitochondrial disorders with optic neuropathy.

Any teaching or reference to SEQ ID NO:34 as disclosed herein in analogyapplies to SEQ ID NO:9.

3. Uses of Polypeptides that Bind hRGM A

Another embodiment of the present application comprises theabove-identified use of isolated proteins or polypeptides thatspecifically bind to at least one epitope of a RGM A protein. Theisolated proteins or polypeptides that specifically bind to at least oneepitope of a RGM A protein are capable of inhibiting binding of RGM A toits receptor Neogenin and/or to bone morphogenetic proteins 2 and 4(BMP-2, BMP-4), in particular antibodies that bind to RGM A orantigen-binding portions or fragments thereof.

Anti-RGM A antibodies of the present invention exhibit a high capacityto reduce or to neutralize RGM A activity, e.g., as assessed by any oneof several in vitro and in vivo assays known in the art or describedbelow.

The present application in particular makes use of neutralizingmonoclonal antibodies against RGM A, which selectively prevent bindingof RGM A to its receptor Neogenin and to bone morphogenetic proteins 2and 4 (BMP-2, BMP-4), and the generation of a neutralizing monoclonalantibody against RGM A, which selectively prevents binding of RGM A toits coreceptors bone morphogenetic proteins 2 and 4 (BMP-2, BMP-4).

In particular, the monoclonal neutralizing antibody of the presentapplication is a human antibody or humanized antibody. The term “humanantibody” refers to antibodies having variable and constant regionscorresponding to, or derived from, human germline immunoglobulinsequences (e.g., see Kabat et al. Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242, 1991). The human antibodies of the presentapplication, however, may include amino acid residues not encoded byhuman germline immunoglobulin sequences (e.g., mutations introduced byrandom or site-specific mutagenesis in vitro or by somatic mutation invivo), for example, in the CDRs and, in particular, CDR3.

In various embodiments, the antibody is a recombinant antibody or amonoclonal antibody. Examples of neutralizing antibodies of the presentapplication are referred to herein as mAb5F9 and mAb8D1 and functionalantibody fragments thereof, and other antibodies and functional antibodyfragments with equivalent properties to mAb5F9 and mAb8D1, such as highaffinity binding to RGM A with low dissociation kinetics and highneutralizing capacity, are intended as part of the present invention.The binding affinity and dissociation rate of an anti-RGM A antibody ofthe present application to an immunogenic RGM A polypeptide or fragmentthereof, may be determined by any method known in the art. For example,the binding affinity can be measured by competitive ELISAs, c RIAs,BIAcore or KinExA technology. The dissociation rate also can be measuredby BIAcore or KinExA technology. The binding affinity and dissociationrate are measured by surface plasmon resonance using, e.g., a BIAcore.

One of the monoclonal antibodies of the present application, the mAb5F9antibody, has at least 90% amino acid sequence identity with a sequencecomprising a heavy chain variable region (VH region) comprising thesequence of SEQ ID NO:9 or 34 and a light chain variable region (VLregion) comprising the sequence of SEQ ID NO:10.

It is also intended that the isolated monoclonal antibodies thatinteract with RGM A of the present application may be a glycosylatedbinding protein wherein the antibody or antigen-binding portion thereofcomprises one or more carbohydrate residues. Nascent in vivo proteinproduction may undergo further processing, known as post-translationalmodification. In particular, sugar (glycosyl) residues may be addedenzymatically, a process known as glycosylation. The resulting proteinsbearing covalently linked oligosaccharide side chains are known asglycosylated proteins or glycoproteins. Protein glycosylation depends onthe amino acid sequence of the protein of interest, as well as the hostcell in which the protein is expressed. Different organisms may producedifferent glycosylation enzymes (eg., glycosyltransferases andglycosidases), and have different substrates (nucleotide sugars)available. Due to such factors, protein glycosylation pattern, andcomposition of glycosyl residues, may differ depending on the hostsystem in which the particular protein is expressed. Glycosyl residuesuseful in the invention may include, but are not limited to, glucose,galactose, mannose, fucose, n-acetylglucosamine and sialic acid.Particularly the glycosylated binding protein comprises glycosylresidues such that the glycosylation pattern is human.

The antibodies as used according to the invention comprise a heavy chainconstant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, IgYor IgD constant region. Furthermore, the antibody can comprise a lightchain constant region, either a kappa light chain constant region or alambda light chain constant region. The antibody comprises a kappa lightchain constant region. Alternatively, the antibody portion can be, forexample, a Fab fragment or a single chain Fv fragment. Replacements ofamino acid residues in the Fc portion to alter antibody effector'sfunction are known in the art (Winter, et al. U.S. Pat. Nos. 5,648,260;5,624,821). The Fc portion of an antibody mediates several importanteffector's functions e.g. cytokine induction, ADCC, phagocytosis,complement dependent cytotoxicity (CDC) and half-life/clearance rate ofantibody and antigen-antibody complexes. In some cases these effector'sfunctions are desirable for therapeutic antibody but in other casesmight be unnecessary or even deleterious, depending on the therapeuticobjectives. Certain human IgG isotypes, particularly IgG1 and IgG3,mediate ADCC and CDC via binding to Fcγ R5 and complement C1q,respectively. Neonatal Fc receptors (FcRn) are the critical componentsdetermining the circulating half-life of antibodies. In still anotherembodiment at least one amino acid residue is replaced in the constantregion of the antibody, for example the Fc region of the antibody, suchthat effector's functions of the antibody are altered.

3. Generation of Anti-hRGM A Antibodies

3.1. General

Antibodies of the invention can be generated by immunization of asuitable host (e.g., vertebrates, including humans, mice, rats, sheep,goats, pigs, cattle, horses, reptiles, fishes, amphibians, and in eggsof birds, reptiles and fish). To generate the antibodies of the presentapplication, the host is immunized with an immunogenic RGM A polypeptideor fragment thereof of the invention. The term “immunization” refersherein to the process of presenting an antigen to an immune repertoirewhether that repertoire exists in a natural genetically unalteredorganism, or a transgenic organism, including those modified to displayan artificial human immune repertoire. Similarly, an “immunogenicpreparation” is a formulation of antigen that contains adjuvants orother additives that would enhance the immunogenicity of the antigen.

Immunization of animals may be done by any method known in the art. See,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: ColdSpring Harbor Press, 1990. Methods for immunizing non-human animals suchas mice, rats, sheep, goats, pigs, cattle and horses are well known inthe art. See, e.g., Harlow and Lane and U.S. Pat. No. 5,994,619. In aparticular embodiment, the RGM A antigen is administered with anadjuvant to stimulate the immune response. Such adjuvants includecomplete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) orISCOM (immunostimulating complexes). Such adjuvants may protect thepolypeptide from rapid dispersal by sequestering it in a local deposit,or they may contain substances that stimulate the host to secretefactors that are chemotactic for macrophages and other components of theimmune system. Particularly, if a polypeptide is being administered, theimmunization schedule will involve two or more administrations of thepolypeptide, spread out over several weeks.

It is contemplated that the animal host is immunized with the antigenassociated with the cell membrane of an intact or disrupted cell andantibodies of the present application are identified by binding to animmunogenic polypeptide of the invention. After immunization of theanimal host with the antigen, antibodies may be obtained from theanimal. The antibody-containing serum is obtained from the animal bybleeding or sacrificing the animal. The serum may be used as it isobtained from the animal, an immunoglobulin fraction may be obtainedfrom the serum, or the antibodies may be purified from the serum. Serumor immunoglobulins obtained in this manner are polyclonal, thus having aheterogeneous array of properties.

3.2 Anti-RGM A Monoclonal Antibodies Using Hybridoma Technology

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In oneembodiment, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, the hybridoma is generated by fusing splenocytesisolated from a mouse immunized with an antigen of the invention withmyeloma cells and then screening the hybridomas resulting from thefusion for hybridoma clones that secrete an antibody able to bind apolypeptide of the invention. Briefly, mice can be immunized with an RGMA antigen. In another embodiment, the RGM A antigen is administered witha adjuvant to stimulate the immune response. Such adjuvants includecomplete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) orISCOM (immunostimulating complexes). Such adjuvants may protect thepolypeptide from rapid dispersal by sequestering it in a local deposit,or they may contain substances that stimulate the host to secretefactors that are chemotactic for macrophages and other components of theimmune system. If a polypeptide is being administered, the immunizationschedule will involve two or more administrations of the polypeptide,spread out over several weeks.

Once an immune response is detected, e.g., antibodies specific for theantigen RGM A are detected in the mouse serum, the mouse spleen isharvested and splenocytes isolated. The splenocytes are then fused bywell-known techniques to any suitable myeloma cells, for example cellsfrom cell line SP20 available from the ATCC. Hybridomas are selected andcloned by limited dilution. The hybridoma clones are then assayed bymethods known in the art for cells that secrete antibodies capable ofbinding RGM A. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

In another embodiment, antibody-producing immortalized hybridomas may beprepared from the immunized animal. After immunization, the animal issacrificed and the splenic B cells are fused to immortalized myelomacells as is well known in the art. See, e.g., Harlow and Lane, supra. Inanother embodiment, the myeloma cells do not secrete immunoglobulinpolypeptides (a non-secretory cell line). After fusion and antibioticselection, the hybridomas are screened using RGM A, or a portionthereof, or a cell expressing RGM A. In another embodiment, the initialscreening is performed using an enzyme-linked immunoassay (ELISA) or aradioimmunoassay (RIA), An ELISA. An example of ELISA screening isprovided in WO 00/37504, herein incorporated by reference.

Anti-RGM A antibody-producing hybridomas are selected, cloned andfurther screened for desirable characteristics, including robusthybridoma growth, high antibody production and desirable antibodycharacteristics, as discussed further below. Hybridomas may be culturedand expanded in vivo in syngeneic animals, in animals that lack animmune system, e.g., nude mice, or in cell culture in vitro. Methods ofselecting, cloning and expanding hybridomas are well known to those ofordinary skill in the art.

In a particular embodiment, the hybridomas are mouse hybridomas, asdescribed above. In another particular embodiment, the hybridomas areproduced in a non-human, non-mouse species such as rats, sheep, pigs,goats, cattle or horses. In another embodiment, the hybridomas are humanhybridomas, in which a human non-secretory myeloma is fused with a humancell expressing an anti-RGM A antibody.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

3.3 Anti-RGM A Monoclonal Antibodies Using SLAM

In another aspect of the invention, recombinant antibodies are generatedfrom single, isolated lymphocytes using a procedure referred to in theart as the selected lymphocyte antibody method (SLAM), as described inU.S. Pat. No. 5,627,052, PCT Publication WO 92/02551 and Babcock, J. S.et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In this method,single cells secreting antibodies of interest, e.g., lymphocytes derivedfrom any one of the immunized animals described above, are screenedusing an antigen-specific hemolytic plaque assay, wherein the antigenRGM A, a subunit of RGM A, or a fragment thereof, is coupled to sheepred blood cells using a linker, such as biotin, and used to identifysingle cells that secrete antibodies with specificity for RGM A.Following identification of antibody-secreting cells of interest, heavy-and light-chain variable region cDNAs are rescued from the cells byreverse transcriptase-PCR and these variable regions can then beexpressed, in the context of appropriate immunoglobulin constant regions(e.g., human constant regions), in mammalian host cells, such as COS orCHO cells. The host cells transfected with the amplified immunoglobulinsequences, derived from in vivo selected lymphocytes, can then undergofurther analysis and selection in vitro, for example by panning thetransfected cells to isolate cells expressing antibodies to RGM A. Theamplified immunoglobulin sequences further can be manipulated in vitro,such as by in vitro affinity maturation methods such as those describedin PCT Publication WO 97/29131 and PCT Publication WO 00/56772.

3.4 Anti-RGM A Monoclonal Antibodies Using Transgenic Animals

In another embodiment of the instant invention, antibodies are producedby immunizing a non-human animal comprising some, or all, of the humanimmunoglobulin locus with an RGM A antigen. In a particular embodiment,the non-human animal is a XENOMOUSE transgenic mouse, an engineeredmouse strain that comprises large fragments of the human immunoglobulinloci and is deficient in mouse antibody production. See, e.g., Green etal. Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771,5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and6,130,364. See also WO 91/10741, published Jul. 25, 1991, WO 94/02602,published Feb. 3, 1994, WO 96/34096 and WO 96/33735, both published Oct.31, 1996, WO 98/16654, published Apr. 23, 1998, WO 98/24893, publishedJun. 11, 1998, WO 98/50433, published Nov. 12, 1998, WO 99/45031,published Sep. 10, 1999, WO 99/53049, published Oct. 21, 1999, WO 0009560, published Feb. 24, 2000 and WO 00/037504, published Jun. 29,2000. The XENOMOUSE transgenic mouse produces an adult-like humanrepertoire of fully human antibodies, and generates antigen-specifichuman Mabs. The XENOMOUSE transgenic mouse contains approximately 80% ofthe human antibody repertoire through introduction of megabase sized,germline configuration YAC fragments of the human heavy chain loci and xlight chain loci. See Mendez et al., Nature Genetics 15: 146-156 (1997),Green and Jakobovits J. Exp. Med. 188: 483-495 (1998), the disclosuresof which are hereby incorporated by reference.

3.5 Anti-RGM A Monoclonal Antibodies Using Recombinant AntibodyLibraries

In vitro methods also can be used to make the antibodies of theinvention, wherein an antibody library is screened to identify anantibody having the desired binding specificity. Methods for suchscreening of recombinant antibody libraries are well known in the artand include methods described in, for example, Ladner et al. U.S. Pat.No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al.PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling etal. PCT Publication No. WO 93/01288; McCafferty et al. PCT PublicationNo. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchset al. (1991) Bio/Technology 9: 1370-1372; Hay et al. (1992) Hum AntibodHybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281;McCafferty et al., Nature (1990) 348: 552-554; Griffiths et al. (1993)EMBO J 12: 725-734; Hawkins et al. (1992) J Mol Biol 226: 889-896;Clackson et al. (1991) Nature 352: 624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9: 1373-1377; Hoogenboomet al. (1991) Nuc Acid Res 19: 4133-4137; and Barbas et al. (1991) PNAS88: 7978-7982, US patent application publication 20030186374, and PCTPublication No. WO 97/29131, the contents of each of which areincorporated herein by reference.

The recombinant antibody library may be from a subject immunized withRGM A, or a portion of RGM A. Alternatively, the recombinant antibodylibrary may be from a naïve subject, i.e., one who has not beenimmunized with RGM A, such as a human antibody library from a humansubject who has not been immunized with human RGM A. Antibodies of theinvention are selected by screening the recombinant antibody librarywith the peptide comprising human RGM A to thereby select thoseantibodies that recognize RGM A. Methods for conducting such screeningand selection are well known in the art, such as described in thereferences in the preceding paragraph. To select antibodies of theinvention having particular binding affinities for hRGM A, such as thosethat dissociate from human RGM A with a particular k_(off) rateconstant, the art-known method of surface plasmon resonance can be usedto select antibodies having the desired k_(off) rate constant. To selectantibodies of the invention having a particular neutralizing activityfor hRGM A, such as those with a particular an IC₅₀, standard methodsknown in the art for assessing the inhibition of hRGM A activity may beused.

In one aspect, the invention pertains to an isolated antibody, or anantigen-binding portion thereof, that binds human RGM A. Particularly,the antibody is a neutralizing antibody. In various embodiments, theantibody is a recombinant antibody or a monoclonal antibody.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182: 41-50 (1995); Ames et al., J. Immunol.Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57: 191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6): 864-869(1992); and Sawai et al., AJRI 34: 26-34 (1995); and Better et al.,Science 240: 1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques, which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203: 46-88 (1991); Shu et al., PNAS 90: 7995-7999 (1993); and Skerra etal., Science 240: 1038-1040 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification of dualspecificity antibodies of the invention. One type of alternativeexpression system is one in which the recombinant antibody library isexpressed as RNA-protein fusions, as described in PCT Publication No. WO98/31700 by Szostak and Roberts, and in Roberts, R. W. and Szostak, J.W. (1997) Proc. Natl. Acad. Sci. USA 94: 12297-12302. In this system, acovalent fusion is created between an mRNA and the peptide or proteinthat it encodes by in vitro translation of synthetic mRNAs that carrypuromycin, a peptidyl acceptor antibiotic, at their 3′ end. Thus, aspecific mRNA can be enriched from a complex mixture of mRNAs (e.g., acombinatorial library) based on the properties of the encoded peptide orprotein, e.g., antibody, or portion thereof, such as binding of theantibody, or portion thereof, to the dual specificity antigen. Nucleicacid sequences encoding antibodies, or portions thereof, recovered fromscreening of such libraries can be expressed by recombinant means asdescribed above (e.g., in mammalian host cells) and, moreover, can besubjected to further affinity maturation by either additional rounds ofscreening of mRNA-peptide fusions in which mutations have beenintroduced into the originally selected sequence(s), or by other methodsfor affinity maturation in vitro of recombinant antibodies, as describedabove.

In another approach the antibodies of the present invention can also begenerated using yeast display methods known in the art. In yeast displaymethods, genetic methods are used to tether antibody domains to theyeast cell wall and display them on the surface of yeast. In particular,such yeast can be utilized to display antigen-binding domains expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Examples of yeast display methods that can be used to make theantibodies of the present invention include those disclosed Wittrup, etal. U.S. Pat. No. 6,699,658 incorporated herein by reference.

4. Production of Particular Recombinant RGM A Antibodies of theInvention

Antibodies of the present invention may be produced by any of a numberof techniques known in the art. For example, expression from host cells,wherein expression vector(s) encoding the heavy and light chains is(are) transfected into a host cell by standard techniques. The variousforms of the term “transfection” are intended to encompass a widevariety of techniques commonly used for the introduction of exogenousDNA into a prokaryotic or eukaryotic host cell, e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection and the like.Although it is possible to express the antibodies of the invention ineither prokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells is of interest, and of most interest in mammalian hostcells, because such eukaryotic cells (and in particular mammalian cells)are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Particular mammalian host cells for expressing the recombinantantibodies of the invention include Chinese Hamster Ovary (CHO cells)(including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc.Natl. Acad. Sci. USA 77: 4216-4220, used with a DHFR selectable marker,e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159: 601-621), NS0 myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more particularly, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding functional fragments of either the light chain and/orthe heavy chain of an antibody of this invention. Recombinant DNAtechnology may also be used to remove some, or all, of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to the antigens of interest. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of theinvention. In addition, bifunctional antibodies may be produced in whichone heavy and one light chain are an antibody of the invention and theother heavy and light chain are specific for an antigen other than theantigens of interest by crosslinking an antibody of the invention to asecond antibody by standard chemical crosslinking methods.

In a particular system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.Still further the invention provides a method of synthesizing arecombinant antibody of the invention by culturing a host cell of theinvention in a suitable culture medium until a recombinant antibody ofthe invention is synthesized. The method can further comprise isolatingthe recombinant antibody from the culture medium.

4.1 Anti RGM A Antibodies

Table 5 is a list of amino acid sequences of VH and VL regions ofparticular anti-hRGM A antibodies of the invention.

TABLE 5 LIST OF AMINO ACID SEQUENCES OF VH AND VL REGIONS OF ANTIhRGM A ANTIBODIES 5F9 AND 8D1

The foregoing isolated anti-RGM A antibody CDR sequences establish anovel family of RGM A binding proteins, isolated in accordance with thisinvention. To generate and to select CDR's of the invention havingparticular RGM A binding and/or neutralizing activity with respect tohRGM A, standard methods known in the art for generating bindingproteins of the present invention and assessing the RGM A binding and/orneutralizing characteristics of those binding protein may be used,including but not limited to those specifically described herein.

4.2 Anti RGM A Chimeric Antibodies

A chimeric antibody is a molecule in which different portions of theantibody are derived from different animal species, such as antibodieshaving a variable region derived from a murine monoclonal antibody and ahuman immunoglobulin constant region. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science 229: 1202(1985); Oi et al., BioTechniques 4: 214 (1986); Gillies et al., (1989)J. Immunol. Methods 125: 191-202; U.S. Pat. Nos. 5,807,715; 4,816,567;and 4,816,397, which are incorporated herein by reference in theirentireties. In addition, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312: 604-608; Takeda et al.,1985, Nature 314: 452-454 which are incorporated herein by reference intheir entireties) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used.

In one embodiment, the chimeric antibodies of the invention are producedby replacing the heavy chain constant region of the murine monoclonalanti human RGM A antibodies described herein with a human IgG1 constantregion.

4.3 Anti RGM A CDR Grafted Antibodies

CDR-grafted antibodies of the invention comprise heavy and light chainvariable region sequences from a human antibody wherein one or more ofthe CDR regions of V_(H) and/or V_(L) are replaced with CDR sequences ofnon-human, as for example murine antibodies of the invention. Aframework sequence from any human antibody may serve as the template forCDR grafting. However, straight chain replacement onto such a frameworkoften leads to some loss of binding affinity to the antigen. The morehomologous a human antibody is to the original murine antibody, the lesslikely the possibility that combining the murine CDRs with the humanframework will introduce distortions in the CDRs that could reduceaffinity. Therefore, it is of particular interest that the humanvariable framework that is chosen to replace the murine variableframework apart from the CDRs have at least a 65% sequence identity withthe murine antibody variable region framework. It is of more particularinterest that the human and murine variable regions apart from the CDRshave at least 70% sequence identify. It is of even more particularinterest that the human and murine variable regions apart from the CDRshave at least 75% sequence identity. It is of most particular interestthat the human and murine variable regions apart from the CDRs have atleast 80% sequence identity. Methods for producing CDR-graftedantibodies are known in the art (Jones et al., Nature 321: 522-525(1986); U.S. Pat. No. 5,225,539). In a specific embodiment the inventionprovides CDR grafted antibodies with V_(H) and/or V_(L) chains asdescribed in Table 6.

TABLE 6 CDR GRAFTED ANTIBODIES SEQ ID Sequence No. Protein region123456789012345678901234567890 35 VH 5F9.1-GL (15) (VH3-48/JH3 FR1)EVQLVESGGGLVQPGGSLRLSCAASGFTFS (16) (VH3-48/JH3 FR2)NYGMNWVRQAPGKGLEWVSMIYYDSSEKHY (17) (VH3-48/JH3 FR3)ADSVKGRFTISRDNAKNSLYLQMNSLRDED (18) (VH3-48/JH3 FR4)TAVYYCARGTTPDYWGQGTMVTVSS 36 VH 5F9.2-GL (15) (VH3-48/JH4 FR1)EVQLVESGGGLVQPGGSLRLSCAASGFTFS (16) (VH3-48/JH4 FR2)NYGMNWVRQAPGKGLEWVSMIYYDSSEKHY (17) (VH3-48/JH4 FR3)ADSVKGRFTISRDNAKNSLYLQMNSLRDED (19) (VH3-48/JH4 FR4)TAVYYCARGTTPDYWGQGTLVTVSS 37 VH 5F9.3-GL (15) (VH3-48/JH6 FR1)EVQLVESGGGLVQPGGSLRLSCAASGFTFS (16) (VH3-48/JH6 FR2)MYGMNWVRQAPGKGLEWVSMIYYDSSEKHY (17) (VH3-48/JH6 FR3)ADSVKGRFTISRDNAKNSLYLQMNSLRDED (20) (VH3-48/JH6 FR4)TAVYYCARGTTPDYWGQGTTVTVSS 38 VH 5F9.4-GL (21) (VH3-33/JH3 FR1)QVQLVESGGGVVQPGRSLRLSCAASGFTFS (22) (VH3-33/JH3 FR2)NYGMNWVRQAPGKGLEWVAMIYYDSSEKHY (23) (VH3-33/JH3 FR3)ADSVKGRFTISRDNSKNTLYLQMNSLRAED (18) (VH3-33/JH3 FR4)TAVYYCARGTTPDYWGQGTMVTVSS 39 VH 5F9.5-GL (21) (VH3-33/JH4 FR1)QVQLVESGGGVVQPGRSLRLSCAASGFTFS (22) (VH3-33/JH4 FR2)NYGMNWVRQAPGKGLEWVAMIYYDSSEKHY (23) (VH3-33/JH4 FR3)ADSVKGRFTISRDNSKNTLYLQMNSLRAED (19) (VH3-33/JH4 FR4)TAVYYCARGTTPDYWGQGTLVTVSS 40 VH 5F9.6-GL (21) (VH3-33/JH6 FR1)QVQLVESGGGVVQPGRSLRLSCAASGFTFS (22) (VH3-33/JH6 FR2)NYGMNWVRQAPGKGLEWVAMIYYDSSEKHY (23) (VH3-33/JH6 FR3)ADSVKGRFTISRDNSKNTLYLQMNSLRAED (20) (VH3-33/JH6 FR4)TAVYYCARGTTPDYWGQGTTVTVSS 41 VH 5F9.7-GL (24) (VH3-23/JH3 FR1)EVQLLESGGGLVQPGGSLRLSCAASGFTFS (25) (VH3-23/JH3 FR2)NYGMNWVRQAPGKGLEWVSMIYYDSSEKHY (26) (VH3-23/JH3 FR3)ADSVKGRFTISRDNSKNTLYLQMNSLRAED (18) (VH3-23/JH3 FR4)TAVYYCAKGTTPDYWGQGTMVTVSS 42 VH 5F9.8-GL (24) (VH3-23/JH4 FR1)EVQLLESGGGLVQPGGSLRLSCAASGFTFS (25) (VH3-23/JH4 FR2)NYGMNWVRQAPGKGLEWVSMIYYDSSEKHY (26) (VH3-23/JH4 FR3)ADSVKGRFTISRDNSKNTLYLQMNSLRAED (19) (VH3-23/JH4 FR4)TAVYYCAKGTTPDYWGQGTLVTVSS 43 VH 5F9.9-GL (24) (VH3-23/JH6 FR1)EVQLLESGGGLVQPGGSLRLSCAASGFTFS (25) (VH3-23/JH6 FR2)NYGMNWVRQAPGKGLEWVSMIYYDSSEKHY (26) (VH3-23/JH6 FR3)ADSVKGRFTISRDNSKNTLYLQMNSLRAED (20) (VH3-23/JH6 FR4)TAVYYCAKGTTPDYWGQGTTVTVSS 44 VL 5F9.1-GL (27) (A18/JK2 FR1)DIVMTQTPSLSLVTPGQPASISCRSSQSLE (28) (A18/JK2 FR2)YSDGYTFLEWYLQKPGQSPQLLIYEVSNRF (29) (A18/JK2 FR3)SGVPDRFSGSGSGTDFTLKISRVEAEDVGV (30) (A18/JK2 FR4)YYCFQATHDPLTFGQGTKLEIKR 45 VL 5F9.2-GL (31) (A17/JK2 FR1)DVVMTQSPLSLPVTLGQPASISCRSSQSLE (32) (A17/JK2 FR2)YSDGYTFLEWFQQRPGQSPRRLIYEVSNRF (33) (A17/JK2 FR3)SGVPDRFSGSGSGTDFTLKISRVEAEDVGV (30) (A17/JK2 FR4)YYCFQATHDPLTFGQGTKLEIKR 46 VL 5F9.3-GL (31) (A17/JK2 FR1)DVVMTQSPLSLPVTLGQPASISCRSSQSLE (28) (A18/JK2 FR2)YSDGYTFLEWYLQKPGQSPQLLIYEVSNRF (29) (A18/JK2 FR3)SGVPDRFSGSGSGTDFTLKISRVEAEDVGV (30) (A18/JK2 FR4)YYCFQATHDPLTFGQGTKLEIKR CDR sequences derived from mAb 5F9 are stated inbold letters. reference is also made to the specific framework sequences(FR1 to FR4) by stating the corresponding SEQ ID Nos (see also Tables 3and 4)4.4 Anti RGM A Humanized Antibodies

Humanized antibodies are antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule. Known humanIg sequences are disclosed, e.g., Kabat et al., Sequences of Proteins ofImmunological Interest, U.S. Dept. Health (1983), each entirelyincorporated herein by reference. Such imported sequences can be used toreduce immunogenicity or reduce, enhance or modify binding, affinity,on-rate, off-rate, avidity, specificity, half-life, or any othersuitable characteristic, as known in the art.

Framework residues in the human framework regions may be substitutedwith the corresponding residue from the CDR donor antibody to alter,particularly improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332: 323(1988), which are incorporated herein by reference in their entireties.)Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Antibodies can be humanized using a variety of techniques known in theart, such as but not limited to those described in Jones et al., Nature321: 522 (1986); Verhoeyen et al., Science 239: 1534 (1988)), Sims etal., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89: 4285(1992); Presta et al., J. Immunol. 151: 2623 (1993), Padlan, MolecularImmunology 28(4/5): 489-498 (1991); Studnicka et al., ProteinEngineering 7(6): 805-814 (1994); Roguska. et al., PNAS 91: 969-973(1994); PCT publication WO 91/09967, PCT/: US98/16280, US96/18978,US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755;WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596,EP 239,400, U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514,5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352,6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539;4,816,567, each entirely incorporated herein by reference, includedreferences cited therein.

5. Further Embodiments of Antibodies of the Invention

5.1 Fusion Antibodies and Immunoadhesins

The present application also describes a fusion antibody orimmunoadhesin that may be made which comprises all or a portion of a RGMA antibody of the present application linked to another polypeptide. Insome embodiments, only the variable region of the RGM A antibody islinked to the polypeptide. In other embodiments, the VH domain of a RGMA antibody of this application is linked to a first polypeptide, whilethe VL domain of the antibody is linked to a second polypeptide thatassociates with the first polypeptide in a manner that permits the VHand VL domains to interact with one another to form an antibody bindingsite. In other embodiments, the VH domain is separated from the VLdomain by a linker that permits the VH and VL domains to interact withone another (see below under Single Chain Antibodies). The VH-linker-VLantibody is then linked to a polypeptide of interest. The fusionantibody is useful to directing a polypeptide to a cell or tissue thatexpresses a RGM A. The polypeptide of interest may be a therapeuticagent, such as a toxin, or may be a diagnostic agent, such as an enzyme;that may be easily visualized, such as horseradish peroxidase. Inaddition, fusion antibodies can be created in which two (or more)single-chain antibodies are linked to one another. This is useful if onewants to create a divalent or polyvalent antibody on a singlepolypeptide chain, or if one wants to create a bispecific antibody.

One embodiment provides a labelled binding protein wherein an antibodyor antibody portion of the present application is derivatized or linkedto another functional molecule (e.g., another peptide or protein). Forexample, a labelled binding protein of the present application can bederived by functionally linking an antibody or antibody portion of thepresent application (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas a nucleic acid, another antibody (e.g., a bispecific antibody or adiabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent,and/or a protein or peptide that can mediate association of the antibodyor antibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag).

Useful detectable agents with which an antibody or antibody portion ofthe present application may be derivatized include fluorescentcompounds. Exemplary fluorescent detectable agents include fluorescein,fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and thelike. An antibody may also be derivatized with detectable enzymes, suchas alkaline phosphatase, horseradish peroxidase, glucose oxidase and thelike. When an antibody is derivatized with a detectable enzyme, it isdetected by adding additional reagents that the enzyme uses to produce adetectable reaction product. For example, when the detectable agenthorseradish peroxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with a nucleic acid,biotin, and detected through indirect measurement of avidin orstreptavidin binding.

5.2 Single Chain Antibodies

The present application includes a single chain antibody (scFv) thatbinds an immunogenic RGM A of the invention. To produce the scFv, VH-and V-encoding DNA is operatively linked to DNA encoding a flexiblelinker, e.g., encoding the amino acid sequence (Gly4-Ser), such that theVH and VL sequences can be expressed as a contiguous single-chainprotein, with the VL and VH regions joined by the flexible linker (seee.g., Bird et al. (1988) Science 242: 423-42 6; Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., 30 Nature(1990) 34 8: 552-554). The single chain antibody may be monovalent, ifonly a single VH and VL are used, bivalent, if two VH and VL are used,or polyvalent, if more than two VH and VL are used. Two of said scFvfragments coupled via a linker are called “diabody” which form is alsoencompassed by the invention.

5.3 Bispecific Antibodies

The present application further includes a bispecific antibody orantigen-binding fragment thereof in which one specificity is for animmunogenic RGM A polypeptide of the present application. For example, abispecific antibody can be generated that specifically binds to animmunogenic RGM A polypeptide of the invention through one bindingdomain and to a second molecule through a second binding domain. Inaddition, a single chain antibody containing more than one VH and VL maybe generated that binds specifically to an immunogenic polypeptide ofthe invention and to another molecule that is associated withattenuating myelin mediated growth cone collapse and inhibition ofneurite outgrowth and sprouting. Such bispecific antibodies can begenerated using techniques that are well known for example, Fanger etal. Immunol Methods 4: 72-81 (1994) and Wright and Harris, 20 (supra).

In some embodiments, the bispecific antibodies are prepared using one ormore of the variable regions from an antibody of the invention. Inanother embodiment, the bispecific antibody is prepared using one ormore CDR regions from said antibody.

5.4 Derivatized and Labeled Antibodies

An antibody or an antigen-binding fragment of the present applicationcan be derivatized or linked to another molecule (e.g., another peptideor protein). In general, the antibody or antigen-binding fragment isderivatized such that binding to an immunogenic polypeptide of theinvention is not affected adversely by the derivatization or labeling.

For example, an antibody or antibody portion of the present applicationcan be functionally linked (by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody (e.g., a bispecific antibody or adiabody), a detection reagent, a cytotoxic agent, a pharmaceuticalagent, and/or a protein or peptide that can mediate association of theantibody or antigen-binding fragment with another molecule (such as astreptavidin core region or a polyhistidine tag). Still further, anantibody or antigen-binding portion thereof may be part of a largerimmunoadhesion molecule, formed by covalent or non-covalent associationof the antibody or antibody portion with one or more other or differentproteins or peptides. Examples of such immunoadhesion molecules includeuse of the streptavidin core region to make a tetrameric scFv molecule(Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) anduse of a cysteine residue, a marker peptide and a C-terminalpolyhistidine tag to make bivalent and biotinylated scFv molecules(Kipriyanov et al. (1994) Molecular Immunology 31:1047-1058). Antibodyportions, such as Fab and F(ab′)₂ fragments, can be prepared from wholeantibodies using conventional techniques, such as papain or pepsindigestion, respectively, of whole antibodies. Moreover, antibodies,antibody portions and immunoadhesion molecules can be obtained usingstandard recombinant DNA techniques.

A derivatized antibody may be produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g. m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

A derivatized antibody may also be a labeled antibody. For instance,detection agents with which an antibody or antibody portion of theinvention may be derivatized are fluorescent compounds, includingfluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. An antibody also may be labeled with enzymesthat are useful for detection, such as horseradish peroxidase,galactosidase, luciferase, alkaline phosphatase, glucoseoxidase and thelike. In embodiments that are labeled with a detectable enzyme, theantibody is detected by adding additional reagents that the enzyme usesto produce a detectable reaction product. For example, horseradishperoxidase with hydrogen peroxide and diaminobenzidine. An antibody alsomay be labeled with biotin, and detected through indirect measurement ofavidin or streptavidin binding. An antibody may also be labeled with apredetermined polypeptide epitope recognized by a secondary reporter(e.g., leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope: tags). An RGM A antibody oran antigen fragment thereof also may be labeled with a radio-labeledamino acid. The radiolabel may be used for both diagnostic andtherapeutic purposes. The radio-labeled RGM A antibody may be useddiagnostically, for example, for determining RGM A receptor levels in asubject. Further, the radio-labeled RGM A antibody may be usedtherapeutically for treating spinal cord injury.

Examples of labels for polypeptides include, but are not limited to, thefollowing radioisotopes or radionucleotides ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, ¹⁵³Sm. A RGM A antibody or an antigen fragmentthereof may also be derivatized with a chemical group such aspolyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrategroup. These groups may be useful to improve the biologicalcharacteristics of the antibody, e.g., to increase serum half-life or toincrease tissue binding. Also, a label for polypeptides can include anucleic acid, for example DNA for detection by PCR, or enhancing geneexpression, or siRNA to suppress gene expression in RGM A-bearing cellsor tissues.

The class and subclass of RGM A antibodies may be determined by anymethod known in the art. In general, the class and subclass of anantibody may be determined using antibodies that are specific for aparticular class and subclass of antibody. Such antibodies are availablecommercially. The class and subclass can be determined by ELISA, WesternBlot as well as other techniques. Alternatively, the class and subclassmay be determined by sequencing all or a portion of the constant domainsof the heavy and/or light chains of the antibodies, comparing theiramino acid sequences to the known amino acid sequences of variousclasses and subclasses of immunoglobulins, and determining the class andsubclass of the antibodies.

5.5 Dual Variable Domain Immunoglobulins

Dual variable domain (DVD) binding proteins or immunoglobulins as usedherein, are binding proteins that comprise two or more antigen bindingsites and are multivalent binding proteins, as for example divalent andtetravalent. The term “multivalent binding protein” is used in thisspecification to denote a binding protein comprising two or more antigenbinding sites. The multivalent binding protein is particularlyengineered to have the two or more antigen binding sites, and isgenerally not a naturally occurring antibody. The term “multispecificbinding protein” refers to a binding protein capable of binding two ormore related or unrelated targets. Such DVDs may be monospecific, i.ecapable of binding one antigen or multispecific, i.e. capable of bindingtwo or more antigens. DVD binding proteins comprising two heavy chainDVD polypeptides and two light chain DVD polypeptides are referred to aDVD Ig. Each half of a DVD Ig comprises a heavy chain DVD polypeptide,and a light chain DVD polypeptide, and two antigen binding sites. Eachbinding site comprises a heavy chain variable domain and a light chainvariable domain with a total of 6 CDRs involved in antigen binding perantigen binding site. DVD binding proteins and methods of making DVDbinding proteins are disclosed in U.S. patent application Ser. No.11/507,050 and incorporated herein by reference. It is intended that thepresent invention comprises a DVD binding protein comprising bindingproteins capable of binding RGM A. Particularly the DVD binding proteinis capable of binding RGM A and a second target. The second target isselected from the group consisting of anti inflammatory MAB activities(IL-1, IL-6, IL-8, IL-11, IL-12, IL-17, IL-18, IL-23, TNF alpha/beta,IFN-beta, gamma, LIF, OSM, CNTF, PF-4, Platelet basic protein (PBP),NAP-2, beta-TG, MIP-1, MCP2/3, RANTES, lymphotactin), oftransport-mediating proteins (insulin receptor, transferrin receptor,thrombin receptor, leptin receptor, LDL receptor) of otherneuroregenerative MABs (NgR, Lingo, p75, CSPG (e.g. NG-2, neurocan,brevican, versican, aggrecan) hyaluronic acid, mAG, tenascin, NI-35,NI-250, IMP, perlecan, neurocan, phosphacan, nogo-A, OMGP, Sema4D, Sema3A, ephrin B3, ephrin A2, ephrin A5, MAG, EphA4, plexin B1, TROY, wnts,ryk rec., BMP-2, BMP-4, BMP-7), of neuroprotective MAB activities (EGF,EGFR, Sema 3), of anti-amyloid beta MABs (e.g. m266, 3D6 (bapineuzumab),anti-globulomer MABs 7C6), of CNS located receptors and transporters(serotonin receptors, dopamine receptors, DAT, Asc-1, GlyT1).

5.6 Dual-Specific Antibodies

The present application also describes “dual-specific antibody”technology. Dual-specific antibodies may serve as agonists, antagonists,or both in different combinations. Dual-specific antibodies areantibodies in which the VH chain binds to a first antigen and the VLchain binds to another antigen as exemplified in WO2008082651.

5.7 Crystallized Antibodies

Another embodiment of the present application provides a crystallizedbinding protein. The term “crystallized” as used herein, refer to anantibody, or antigen binding portion thereof, that exists in the form ofa crystal. Crystals are one form of the solid state of matter, which isdistinct from other forms such as the amorphous solid state or theliquid crystalline state. Crystals are composed of regular, repeating,three-dimensional arrays of atoms, ions, molecules (e.g., proteins suchas antibodies), or molecular assemblies (e.g., antigen/antibodycomplexes). These three-dimensional arrays are arranged according tospecific mathematical relationships that are well understood in thefield. The fundamental unit, or building block, that is repeated in acrystal is called the asymmetric unit. Repetition of the asymmetric unitin an arrangement that conforms to a given, well-definedcrystallographic symmetry provides the “unit cell” of the crystal.Repetition of the unit cell by regular translations in all threedimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett,Crystallization of Nucleic Acids and Proteins, a Practical Approach,2^(nd) ed., pp. 20 1-16, Oxford University Press, New York, N.Y.,(1999).

Particularly the present application describes crystals of whole RGM Aantibodies and fragments thereof as disclosed herein, and formulationsand compositions comprising such crystals. In one embodiment thecrystallized binding protein has a greater half-life in vivo than thesoluble counterpart of the binding protein. In another embodiment thebinding protein retains biological activity after crystallization.

Crystallized binding protein of the invention may be produced accordingmethods known in the art and as disclosed in WO 02072636, incorporatedherein by reference.

5.8 Glycosylated Antibodies

Another embodiment of the invention provides a glycosylated bindingprotein wherein the antibody or antigen-binding portion thereofcomprises one or more carbohydrate residues. Nascent in vivo proteinproduction may undergo further processing, known as post-translationalmodification. In particular, sugar (glycosyl) residues may be addedenzymatically, a process known as glycosylation. The resulting proteinsbearing covalently linked oligosaccharide side chains are known asglycosylated proteins or glycoproteins. Antibodies are glycoproteinswith one or more carbohydrate residues in the Fc domain, as well as thevariable domain. Carbohydrate residues in the Fc domain have importanteffect on the effector function of the Fc domain, with minimal effect onantigen binding or half-life of the antibody (R. Jefferis, Biotechnol.Prog. 21 (2005), pp. 11-16). In contrast, glycosylation of the variabledomain may have an effect on the antigen binding activity of theantibody. Glycosylation in the variable domain may have a negativeeffect on antibody binding affinity, likely due to steric hindrance (Co,M. S., et al., Mol. Immunol. (1993) 30:1361-1367), or result inincreased affinity for the antigen (Wallick, S. C., et al., Exp. Med.(1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991) 10:2717 2723).

One aspect of the present invention is directed to generatingglycosylation site mutants in which the O- or N-linked glycosylationsite of the binding protein has been mutated. One skilled in the art cangenerate such mutants using standard well-known technologies.Glycosylation site mutants that retain the biological activity but haveincreased or decreased binding activity are another object of thepresent invention.

In still another embodiment, the glycosylation of the antibody orantigen-binding portion of the invention is modified. For example, anaglycoslated antibody can be made (i.e., the antibody lacksglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region glycosylation sites to thereby eliminateglycosylation at that site. Such a glycosylation may increase theaffinity of the antibody for antigen. Such an approach is described infurther detail in PCT Publication WO2003016466A2, and U.S. Pat. Nos.5,714,350 and 6,350,861, each of which is incorporated herein byreference in its entirety.

Additionally or alternatively, a modified antibody of the invention canbe made that has an altered type of glycosylation, such as ahypofucosylated antibody having reduced amounts of fucosyl residues oran antibody having increased bisecting GlcNAc structures. Such alteredglycosylation patterns have been demonstrated to increase the ADCCability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields, R. L. et al. (2002) J. Biol. Chem. 277: 26733-26740; Umana etal. (1999) Nat. Biotech. 17: 176-1, as well as, European Patent No: EP1,176,195; PCT Publications WO 03/035835; WO 99/54342 80, each of whichis incorporated herein by reference in its entirety.

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (eg.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful in the invention may include, butare not limited to, glucose, galactose, mannose, fucose,n-acetylglucosamine and sialic acid. Particularly the glycosylatedbinding protein comprises glycosyl residues such that the glycosylationpattern is human.

It is known to those skilled in the art that differing proteinglycosylation may result in differing protein characteristics. Forinstance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the yeastendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vivo after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream. Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using techniques known in the art apractitioner may generate antibodies or antigen-binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (U.S. patentapplications 20040018590 and 20020137134 and PCT publicationWO2005100584 A2).

Further, it will be appreciated by one skilled in the art that a proteinof interest may be expressed using a library of host cells geneticallyengineered to express various glycosylation enzymes, such that memberhost cells of the library produce the protein of interest with variantglycosylation patterns. A practitioner may then select and isolate theprotein of interest with particular novel glycosylation patterns.Particularly, the protein having a particularly selected novelglycosylation pattern exhibits improved or altered biological properties

5.9 Anti-Idiotypic Antibodies

In addition to the binding proteins, the present invention is alsodirected to an anti-idiotypic (anti-Id) antibody specific for suchbinding proteins of the invention. An anti-Id antibody is an antibody,which recognizes unique determinants generally associated with theantigen-binding region of another antibody. The anti-Id can be preparedby immunizing an animal with the binding protein or a CDR containingregion thereof. The immunized animal will recognize, and respond to theidiotypic determinants of the immunizing antibody and produce an anti-Idantibody. The anti-Id antibody may also be used as an “immunogen” toinduce an immune response in yet another animal, producing a so-calledanti-anti-Id antibody.

6. Pharmaceutical Compositions

The invention also provides pharmaceutical compositions comprising anantibody, or antigen-binding portion thereof, of the invention and apharmaceutically acceptable carrier. The pharmaceutical compositionscomprising antibodies of the invention are for use in, but not limitedto, diagnosing, detecting, or monitoring a disorder, in preventing,treating, managing, or ameliorating of a disorder or one or moresymptoms thereof, and/or in research. In a specific embodiment, acomposition comprises one or more antibodies of the invention. Inanother embodiment, the pharmaceutical composition comprises one or moreantibodies of the invention and one or more prophylactic or therapeuticagents other than antibodies of the invention for treating a disorder inwhich RGM A activity is detrimental. Particularly, the prophylactic ortherapeutic agents known to be useful for or having been or currentlybeing used in the prevention, treatment, management, or amelioration ofa disorder or one or more symptoms thereof. In accordance with theseembodiments, the composition may further comprise of a carrier, diluentor excipient.

The antibodies and antibody-portions of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody or antibody portion of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will beof particular interest to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition. Pharmaceutically acceptable carriers may further compriseminor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, which enhance the shelf life oreffectiveness of the antibody or antibody portion.

Various delivery systems are known and can be used to administer one ormore antibodies of the invention or the combination of one or moreantibodies of the invention and a prophylactic agent or therapeuticagent useful for preventing, managing, treating, or ameliorating adisorder or one or more symptoms thereof, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or antibody fragment, receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)),construction of a nucleic acid as part of a retroviral or other vector,etc. Methods of administering a prophylactic or therapeutic agent of theinvention include, but are not limited to, parenteral administration(e.g., intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidural administration, intratumoral administration, andmucosal administration (e.g., intranasal and oral routes). In addition,pulmonary administration can be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064,5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, eachof which is incorporated herein by reference their entireties. In oneembodiment, an antibody of the invention, combination therapy, or acomposition of the invention is administered using Alkermes AIR®pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).In a specific embodiment, prophylactic or therapeutic agents of theinvention are administered intramuscularly, intravenously,intratumorally, orally, intranasally, pulmonary, or subcutaneously. Theprophylactic or therapeutic agents may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents of the invention locally to the areain need of treatment; this may be achieved by, for example, and not byway of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., Tisseel®), or collagen matrices. In oneembodiment, an effective amount of one or more antibodies of theinvention antagonists is administered locally to the affected area to asubject to prevent, treat, manage, and/or ameliorate a disorder or asymptom thereof. In another embodiment, an effective amount of one ormore antibodies of the invention is administered locally to the affectedarea in combination with an effective amount of one or more therapies(e.g., one or more prophylactic or therapeutic agents) other than anantibody of the invention of a subject to prevent, treat, manage, and/orameliorate a disorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent can bedelivered in a controlled release or sustained release system. In oneembodiment, a pump may be used to achieve controlled or sustainedrelease (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14: 20; Buchwald et al., 1980, Surgery 88: 507; Saudek et al., 1989, N.Engl. J. Med. 321: 574). In another embodiment, polymeric materials canbe used to achieve controlled or sustained release of the therapies ofthe invention (see e.g., Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.,Macromol. Sci. Rev. Macromol. Chem. 23: 61; see also Levy et al., 1985,Science 228: 190; During et al., 1989, Ann. Neurol. 25: 351; Howard etal., 1989, J. Neurosurg. 7 1: 105); U.S. Pat. No. 5,679,377; U.S. Pat.No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S.Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT PublicationNo. WO 99/20253. Examples of polymers used in sustained releaseformulations include, but are not limited to, poly(2-hydroxy ethylmethacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In a particularembodiment, the polymer used in a sustained release formulation isinert, free of leachable impurities, stable on storage, sterile, andbiodegradable. In yet another embodiment, a controlled or sustainedrelease system can be placed in proximity of the prophylactic ortherapeutic target, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer (1990,Science 249: 1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698,Ning et al., 1996, “Intratumoral Radioimmunotheraphy of a Human ColonCancer Xenograft Using a Sustained-Release Gel,” Radiotherapy &Oncology39: 179-189, Song et al., 1995, “Antibody Mediated Lung Targeting ofLong-Circulating Emulsions,” PDA Journal of Pharmaceutical Science&Technology 50: 372-397, Cleek et al., 1997, “Biodegradable PolymericCarriers for a bFGF Antibody for Cardiovascular Application,” Pro.Int'l. Symp. Control. Rel. Bioact. Mater. 24: 853-854, and Lam et al.,1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibodyfor Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in theirentireties.

In a specific embodiment, where the composition of the invention is anucleic acid encoding a prophylactic or therapeutic agent, the nucleicacid can be administered in vivo to promote expression of its encodedprophylactic or therapeutic agent, by constructing it as part of anappropriate nucleic acid expression vector and administering it so thatit becomes intracellular, e.g., by use of a retroviral vector (see U.S.Pat. No. 4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88: 1864-1868). Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression byhomologous recombination.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection.

If the compositions of the invention are to be administered topically,the compositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.(1995). For non-sprayable topical dosage forms, viscous to semi-solid orsolid forms comprising a carrier or one or more excipients compatiblewith topical application and having a dynamic viscosity particularlygreater than water are typically employed. Suitable formulationsinclude, without limitation, solutions, suspensions, emulsions, creams,ointments, powders, liniments, salves, and the like, which are, ifdesired, sterilized or mixed with auxiliary agents (e.g., preservatives,stabilizers, wetting agents, buffers, or salts) for influencing variousproperties, such as, for example, osmotic pressure. Other suitabletopical dosage forms include sprayable aerosol preparations wherein theactive ingredient, particularly in combination with a solid or liquidinert carrier, is packaged in a mixture with a pressurized volatile(e.g., a gaseous propellant, such as freon) or in a squeeze bottle.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art.

If the method of the invention comprises intranasal administration of acomposition, the composition can be formulated in an aerosol form,spray, mist or in the form of drops. In particular, prophylactic ortherapeutic agents for use according to the present invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane, carbon dioxide or other suitable gas). Inthe case of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

If the method of the invention comprises oral administration,compositions can be formulated orally in the form of tablets, capsules,cachets, gelcaps, solutions, suspensions, and the like. Tablets orcapsules can be prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinised maizestarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose, or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well-known in the art. Liquid preparations for oraladministration may take the form of, but not limited to, solutions,syrups or suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated for slow release, controlledrelease, or sustained release of a prophylactic or therapeutic agent(s).

The method of the invention may comprise pulmonary administration, e.g.,by use of an inhaler or nebulizer, of a composition formulated with anaerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320,5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078;and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO98/31346, and WO 99/66903, each of which is incorporated herein byreference their entireties. In a specific embodiment, an antibody of theinvention, combination therapy, and/or composition of the invention isadministered using Alkermes AIR® pulmonary drug delivery technology(Alkermes, Inc., Cambridge, Mass.).

The method of the invention may comprise administration of a compositionformulated for parenteral administration by injection (e.g., by bolusinjection or continuous infusion). Formulations for injection may bepresented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use. The methods ofthe invention may additionally comprise of administration ofcompositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

The methods of the invention encompass administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, compositioncan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

Another embodiment provides that one or more of the prophylactic ortherapeutic agents, or pharmaceutical compositions of the invention ispackaged in a hermetically sealed container such as an ampoule orsachette indicating the quantity of the agent. In one embodiment, one ormore of the prophylactic or therapeutic agents, or pharmaceuticalcompositions of the invention is supplied as a dry sterilizedlyophilized powder or water free concentrate in a hermetically sealedcontainer and can be reconstituted (e.g., with water or saline) to theappropriate concentration for administration to a subject. Particularly,one or more of the prophylactic or therapeutic agents or pharmaceuticalcompositions of the invention is supplied as a dry sterile lyophilizedpowder in a hermetically sealed container at a unit dosage of at least 5mg, more particularly at least 10 mg, at least 15 mg, at least 25 mg, atleast 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least100 mg. The lyophilized prophylactic or therapeutic agents orpharmaceutical compositions of the invention should be stored at between2° C. and 8° C. in its original container and the prophylactic ortherapeutic agents, or pharmaceutical compositions of the inventionshould be administered within 1 week, particularly within 5 days, within72 hours, within 48 hours, within 24 hours, within 12 hours, within 6hours, within 5 hours, within 3 hours, or within 1 hour after beingreconstituted. In an alternative embodiment, one or more of theprophylactic or therapeutic agents or pharmaceutical compositions of theinvention is supplied in liquid form in a hermetically sealed containerindicating the quantity and concentration of the agent. Particularly,the liquid form of the administered composition is supplied in ahermetically sealed container at least 0.25 mg/ml, more particularly atleast 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml,at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml. Theliquid form should be stored at between 2° C. and 8° C. in its originalcontainer.

The antibodies and antibody-portions of the invention can beincorporated into a pharmaceutical composition suitable for parenteraladministration. Particularly, the antibody or antibody-portions will beprepared as an injectable solution containing 0.1-250 mg/ml antibody.The injectable solution can be composed of either a liquid orlyophilized dosage form in a flint or amber vial, ampoule or pre-filledsyringe. The buffer can be L-histidine (1-50 mM), optimally 5-10 mM, atpH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but arenot limited to, sodium succinate, sodium citrate, sodium phosphate orpotassium phosphate. Sodium chloride can be used to modify the toxicityof the solution at a concentration of 0-300 mM (optimally 150 mM for aliquid dosage form). Cryoprotectants can be included for a lyophilizeddosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Othersuitable cryoprotectants include trehalose and lactose. Bulking agentscan be included for a lyophilized dosage form, principally 1-10%mannitol (optimally 2-4%). Stabilizers can be used in both liquid andlyophilized dosage forms, principally 1-50 mM L-Methionine (optimally5-10 mM). Other suitable bulking agents include glycine, arginine, canbe included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%).Additional surfactants include but are not limited to polysorbate 20 andBRIJ surfactants. The pharmaceutical composition comprising theantibodies and antibody-portions of the invention prepared as aninjectable solution for parenteral administration, can further comprisean agent useful as an adjuvant, such as those used to increase theabsorption, or dispersion of a therapeutic protein (e.g., antibody). Aparticularly useful adjuvant is hyaluronidase, such as Hylenex®(recombinant human hyaluronidase). Addition of hyaluronidase in theinjectable solution improves human bioavailability following parenteraladministration, particularly subcutaneous administration. It also allowsfor greater injection site volumes (i.e. greater than 1 ml) with lesspain and discomfort, and minimum incidence of injection site reactions.(see WO2004078140, US2006104968 incorporated herein by reference).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The particular form depends on the intended mode of administration andtherapeutic application. Typical particular compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Theparticular mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a particularembodiment, the antibody is administered by intravenous infusion orinjection. In another particular embodiment, the antibody isadministered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the particular methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding, in the composition, an agent that delays absorption, forexample, monostearate salts and gelatin.

The antibodies and antibody-portions of the present invention can beadministered by a variety of methods known in the art, although for manytherapeutic applications, the particular route/mode of administration issubcutaneous injection, intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

In certain embodiments, an antibody or antibody portion of the inventionmay be orally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion ofthe invention is coformulated with and/or coadministered with one ormore additional therapeutic agents that are useful for treatingdisorders in which RGM A activity is detrimental. For example, ananti-RGM A antibody or antibody portion of the invention may becoformulated and/or coadministered with one or more additionalantibodies that bind other targets (e.g., antibodies that bind cytokinesor that bind cell surface molecules). Furthermore, one or moreantibodies of the invention may be used in combination with two or moreof the foregoing therapeutic agents. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies.

In certain embodiments, an antibody to RGM A or fragment thereof islinked to a half-life extending vehicle known in the art. Such vehiclesinclude, but are not limited to, the Fc domain, polyethylene glycol, anddextran. Such vehicles are described, e.g., in U.S. application Ser. No.09/428,082 and published PCT Application No. WO 99/25044, which arehereby incorporated by reference for any purpose.

In a specific embodiment, nucleic acid sequences comprising nucleotidesequences encoding an antibody of the invention or another prophylacticor therapeutic agent of the invention are administered to treat,prevent, manage, or ameliorate a disorder or one or more symptomsthereof by way of gene therapy. Gene therapy refers to therapy performedby the administration to a subject of an expressed or expressiblenucleic acid. In this embodiment of the invention, the nucleic acidsproduce their encoded antibody or prophylactic or therapeutic agent ofthe invention that mediates a prophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. For general reviews of the methodsof gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3: 87-95; Tolstoshev, 1993, Ann.Rev. Pharmacol. Toxicol. 32: 573-596; Mulligan, Science 260: 926-932(1993); and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191-217;May, 1993, TIBTECH 11(5): 155-215. Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons,NY (1993); and Kriegler, Gene Transfer and Expression, A LaboratoryManual, Stockton Press, NY (1990). Detailed description of variousmethods of gene therapy are disclosed in US20050042664 A1 which isincorporated herein by reference.

Any neuroprotective agent being it an antioxidant, a radical scavengers,an anti-convulsive drug like Phenyloin or the anemia drug Erythropoetinis suitable for a combinatorial therapy with pro-regenerative RGM Aantibodies thereby extending the usually very short therapeutictreatment window of the neuroprotectants.

The antibodies, and antibody portions of the invention can be used totreat humans suffering from such diseases.

It should be understood that the antibodies of the invention or antigenbinding portion thereof can be used alone or in combination with anadditional agent, e.g., a therapeutic agent, said additional agent beingselected by the skilled artisan for its intended purpose. For example,the additional agent can be a therapeutic agent art-recognized as beinguseful to treat the disease or condition being treated by the antibodyof the present invention. The additional agent also can be an agent thatimparts a beneficial attribute to the therapeutic composition e.g., anagent, which effects the viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limited. The combinations, which arepart of this invention, can be the antibodies of the present inventionand at least one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

Non-limiting examples of therapeutic agents for multiple sclerosis withwhich an antibody, or antibody portion, of the invention can be combinedinclude the following: corticosteroids; prednisolone;methylprednisolone; azathioprine; cyclophosphamide; cyclosporine;methotrexate; 4-aminopyridine; tizanidine; interferon-β1a (AVONEX;Biogen); interferon-β1b (BETASERON; Chiron/Berlex); interferon α-n3)(Interferon Sciences/Fujimoto), interferon-α (Alfa Wassermann/J&J),interferon β1A-IF (Serono/Inhale Therapeutics), Peginterferon α 2b(Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE; TevaPharmaceutical Industries, Inc.); hyperbaric oxygen; intravenousimmunoglobulin; cladribine; antibodies to or antagonists of other humancytokines or growth factors and their receptors, for example, TNF, LT,IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18, EMAP-II,GM-CSF, FGF, and PDGF. Antibodies of the invention, or antigen bindingportions thereof, can be combined with antibodies to cell surfacemolecules such as CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30,CD40, CD45, CD69, CD80, CD86, CD90 or their ligands. The antibodies ofthe invention, or antigen binding portions thereof, may also be combinedwith agents, such as methotrexate, cyclosporine, FK506, rapamycin,mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,corticosteroids such as prednisolone, phosphodiesterase inhibitors,adenosine agonists, antithrombotic agents, complement inhibitors,adrenergic agents, agents which interfere with signalling byproinflammatory cytokines such as TNFα or IL-1 (e.g. IRAK, NIK, IKK, p38or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TACEinhibitors, T-cell signaling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNFreceptors, sIL-1RI, sIL-1RII, sIL-6R) and antiinflammatory cytokines(e.g. IL-4, IL-10, IL-13 and TGFβ).

Particular examples of therapeutic agents for multiple sclerosis inwhich the antibody or antigen binding portion thereof can be combined toinclude interferon-β, for example, IFNβ1a and IFNβ1b; copaxone,corticosteroids, caspase inhibitors, for example inhibitors ofcaspase-1, IL-1 inhibitors, TNF inhibitors, and antibodies to CD40ligand and CD80.

The antibodies of the invention, or antigen binding portions thereof,may also be combined with agents, such as alemtuzumab, dronabinol,Unimed, daclizumab, mitoxantrone, xaliproden hydrochloride, fampridine,glatiramer acetate, natalizumab, sinnabidol, a-immunokine NNSO3,ABR-215062, AnergiX.MS, chemokine receptor antagonists, BBR-2778,calagualine, CPI-1189, LEM (liposome encapsulated mitoxantrone), THC.CBD(cannabinoid agonist) MBP-8298, mesopram (PDE4 inhibitor), MNA-715,anti-IL-6 receptor antibody, neurovax, pirfenidone allotrap 1258(RDP-1258), sTNF-R1, talampanel, teriflunomide, TGF-beta2, tiplimotide,VLA-4 antagonists (for example, TR-14035, VLA4 Ultrahaler,Antegran-ELAN/Biogen), interferon gamma antagonists, IL-4 agonists.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may be determined by a person skilled in the art andmay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody, or antibody portion, are outweighedby the therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.1-20 mg/kg, more particularly 1-10 mg/kg. It is to benoted that dosage values may vary with the type and severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein. Having now described the present invention in detail,the same will be more clearly understood by reference to the followingexamples, which are included for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES Methods

The following methods describe in detail the experimental proceduresused in the Examples section.

(i) Direct binding ELISA plates were coated with hRGM A (R&D) at aconcentration of 2 μg/mL in Carbonate buffer. The wells were thenblocked with 2% Blocking solution (Bio-Rad) for 1 hour at roomtemperature. Biotinylated antibodies were serially diluted with a 1:5dilution factor in 0.1% BSA/PBS down the plate and incubated for 1 hourat room temperature. The detection reagent was a 1:10,000 dilution ofstreptavidin-HRP in 0.1% BSA/PBS. Detection was done with a TMB reagent,which was stopped with 2NH₂SO₄ and OD was read at 450 nM.

(ii) FACS analysis. Stable transfectants of HEK293 cells overexpressinghRGM A or BAF3 cells overexpressing ratRGM A were subjected to stainingwith unlabelled 5F9 or 8D1 MABs for more than 15 minutes at 4° in 0.1%BSA/PBS buffer. Detection was carried out with a mouse anti-rat IgG PEantibody.

(iii) Solid phase ELISA assays for evaluating MAB 5F9 in hRGM A-Neogeninbinding assays.

ELISA plates (Immuno Plate Cert. Maxi Sorb. F96 NUNC, 439454) werecoated for 1 h at 37° C. with a concentration of 2.5 μg/ml of theextracellular domain of the His-tagged human Neogenin protein(concentration of stock solution: 30 μg/ml). After the incubation,unbound Neogenin was removed in 3 separate wash steps with PBScontaining 0.02% Tween 20. Blocking of the Neogenin-coated plates wasdone by adding 200 μl per well of a 3% Bovine serum albumin (BSA), PBS,Tween 20 (0.02%) blocking solution. After incubation for 1 h at 37° C.,the blocking solution was removed and RGM A fragments or full lengthprotein, conjugated with a human fc tag, with or without antibody, wasadded. In some experiments antibodies were preincubated with thefc-conjugated hRGM A proteins for 1 h at room temperature. TheNeogenin-coated plates were incubated with hRGM A with or withoutantibodies for 1 h at 37° C. After 3 wash steps with PBS-Tween 20(0.02%), plates were incubated with a Biotin-labeled anti-human fcantibody (1 mg/ml, diluted 1:200 in PBS containing 0.6% BSA, 0.02% Tween20), Jackson ImmunoResearch catalog no: 709-065-149, for 1 h at 37° C.Unbound antibody was removed by 3 wash steps with PBS-Tween 20 (0.02%).To visualize binding of the Biotin-labeled anti-fc antibody, a complexconsisting of Streptavidin-Peroxidase (Roche, cat.#11089153001), diluted1:5000 with PBS containing 0.6% BSA, 0.02% Tween 20 was added, followedby incubation at 37° C. for 1 h. Unbound Peroxidase-complex was removedin 3 subsequent wash steps (PBS-Tween 20 (0.02%) before adding thePeroxidase substrate (Immuno Pure TMB, Pierce #34021). The substratereaction was stopped 1-30 min after its addition to the wells by 2.5 MH₂SO₄. Plates were analyzed (OD determination) at a wave length of 450nm using an Anthos photometer.

(iv) Solid phase ELISA assays for evaluating MAB 5F9 in hRGM A-BMP-4binding assays.

ELISA plates (Immuno Plate Cert. Maxi Sorb. F96 NUNC, 439454) werecoated for 1 h at 37° C. with a solution containing a concentration of2.5 μg/ml of recombinant human BMP-4 protein (R&D Systems, #314-BP, Lot# BEM316061). After the incubation, unbound BMP-4 was removed in 3separate wash steps with PBS containing 0.02% Tween 20. Blocking of theBMP-4 coated plates was done by adding 200 μl per well of a 3% Bovineserum albumin (BSA), PBS, Tween 20 (0.02%) blocking solution. Afterincubation for 1 h at 37° C., the blocking solution was removed and RGMA fragments or full length protein, conjugated with a human fc tag, withor without antibody, was added. In some experiments antibodies werepreincubated with the fc-conjugated hRGM A proteins for 1 h at roomtemperature. The BMP-4 coated plates were incubated with hRGM A with orwithout antibodies for 1 h at 37° C. After 3 wash steps with PBS-Tween20 (0.02%), plates were incubated with a Biotin-labeled anti-human fcantibody (1 mg/ml, diluted 1:200 in PBS containing 0.6% BSA, 0.02% Tween20), Jackson ImmunoResearch catalog no: 709,065-149, for 1 h at 37° C.Unbound antibody was removed by 3 wash steps with PBS-Tween 20 (0.02%).To visualize binding of the Biotin-labeled anti-fc antibody, a complexconsisting of Streptavidin-Peroxidase (Roche, cat.#11089153001), diluted1:5000 with PBS containing 0.6% BSA, 0.02% Tween 20 was added, followedby incubation at 37° C. for 1 h. Unbound Peroxidase-complex was removedin 3 subsequent wash steps (PBS-Tween 20 (0.02%) before adding thePeroxidase substrate (Immuno Pure TMB, Pierce #34021). The substratereaction was stopped 1-30 min after its addition to the wells by 2.5 MH₂SO₄. Plates were analyzed (OD determination) at a wave length of 450nm using an Anthos photometer.

(v) Solid phase ELISA assays for evaluating MAB 5F9 in hRGM A-BMP-2binding assays.

ELISA plates (Immuno Plate Cert. Maxi Sorb. F96 NUNC, 439454) werecoated for 1 h at 37° C. with a solution containing a concentration of2.5 μg/ml of recombinant human BMP-2 protein (R&D Systems, #355-BM, Lot# MSA04). After the incubation, unbound BMP-2 was removed in 3 separatewash steps with PBS containing 0.02% Tween 20. Blocking of the BMP-2coated plates was done by adding 200 μA per well of a 3% Bovine serumalbumin (BSA), PBS, Tween 20 (0.02%) blocking solution. After incubationfor 1 h at 37° C., the blocking solution was removed and RGM A fragmentsor full length protein, conjugated with a human fc tag, with or withoutantibody, was added. In some experiments antibodies were preincubatedwith the fc-conjugated hRGM A proteins for 1 h at room temperature. TheBMP-2 coated plates were incubated with hRGM A with or withoutantibodies for 1 h at 37° C. After 3 wash steps with PBS-Tween 20(0.02%), plates were incubated with a Biotin-labeled anti-human fcantibody (1 mg/ml, diluted 1:200 in PBS containing 0.6% BSA, 0.02% Tween20), Jackson ImmunoResearch catalog no: 709, 065-149, for 1 h at 37° C.Unbound antibody was removed by 3 wash steps with PBS-Tween 20 (0.02%).To visualize binding of the Biotin-labeled anti-fc antibody, a complexconsisting of Streptavidin-Peroxidase (Roche, cat.#11089153001), diluted1:5000 with PBS containing 0.6% BSA, 0.02% Tween 20 was added, followedby incubation at 37° C. for 1 h. Unbound Peroxidase-complex was removedin 3 subsequent wash steps (PBS-Tween 20 (0.02%) before adding thePeroxidase substrate (Immuno Pure TMB, Pierce #34021). The substratereaction was stopped 1-30 min after its addition to the wells by 2.5 MH₂SO₄. Plates were analyzed (OD determination) at a wave length of 450nm using an Anthos photometer.

(vi) Ntera-2 cell culture

Human Ntera-2 cells were obtained from the German Collection ofMicroorganisms and Cell Cultures (DMSZ, Braunschweig). Frozen stocks ofundifferentiated Ntera-2 cells were thawed in DMEM medium containing 10%fetal bovine serum (FBS; JRH Bioscience, Kansas, USA) and 5% horse serum(HS; Sigma, Germany). Cells were grown in culture flasks (Greiner,Germany) until they reached confluence of 80%.

For neuronal differentiation, Ntera-2 cells were seeded at a density of2.5×10⁶ cells/175 cm² in differentiation medium (DMEM medium containing10% FBS, 5% HS, 1% penicillin-streptomycin, retinoic acid 10 μM). Cellswere differentiated for 3 weeks and the medium was exchanged twice aweek.

After differentiation, cells were detached with trypsin-EDTA and splitat a ratio of 1:6. 48 h later neuronal cells were separated by tappingfrom the underlying cells. Dislodged cells were transferred foraggregation in new medium into new shaking culture flasks (Corning,USA). Differentiated Ntera-2 cells were allowed to aggregate undersmooth horizontal shaking conditions at 37° C., for 24 h in Neurobasalmedium (Gibco) supplemented with B27 (Gibco), glutamine (Gibco) andpenicillin-streptomycin. Ntera-2 aggregates were seeded at a density ofapproximately 20-30 aggregates per cover slip in 24-well plates. Thepoly-lysine precoated cover slips were coated with laminin (20 μg/ml,Sigma) and with the recombinant fc-coupled human RGM A fragment #786(amino acids 47-168) at a concentration of 10 μg/ml. After seeding,cultures were treated with the 5F9 MAB, added at three differentconcentrations (0.1 μg/ml; 1 μg/ml; 10 μg/ml) to the culture medium andwere further incubated for 24 h at 37° C. in Neurobasal medium.Aggregates were then fixed in 4% paraformaldehyde (2 h, roomtemperature) and permeabilized by addition of 0.1% Triton X-100 in PBS(20 min. room temperature). For fluorescent staining cultures wereblocked with PBS containing 1% BSA for 1 h at room temperature. Afterblocking Ntera cells were incubated with a mouse monoclonal antibodyagainst β-tubulin isotype 3 (clone SDL3D10, Sigma # T8660) for 2 h atroom temperature. Unbound antibody was removed by 3 different wash steps(5-15 min each) and Ntera cells were incubated with a Cy-3 conjugatedDonkey anti-mouse antibody (Jackson ImmunoResearch Lot 62597), diluted1:350 fold in PBS/0.5% BSA and 0.5 μg/ml bisbenzimide. Ater a 1 hourincubation, cultures were washed 3 times to remove unbound secondaryantibody. For fluorescence microscopy, coverslips were embedded inFluoromount G (Southern Biotech, Eching).

Images of Ntera-2 aggregates were acquired using a Zeiss Axiovert 200fluorescence microscope and the outgrowth of the cultures wasautomatically analyzed using an in-house image acquisition and analysissystem. Automatic analysis of outgrowth was done with Image Pro Plus 4.5and the statistical analysis of the data was performed with Graph PadPrism 4. Outgrowth was normalized to control cultures grown in theabsence of the human RGM A fragment #786.

(vii) SH-SY5Y culture.

SH-SY5Y cells (ATCC, CRL-2266) are human neuroblastoma cells derivedfrom a metastatic brain tumor. These cells were grown in a mediumconsisting of 50% Earle's Balanced Salt Solution (Invitrogen LifeTechnologies, Cat. #24010-043) and 50% F12 (Ham) Nutrient Mix+GlutaMAX-1(Invitrogen Life Technologies, Cat. #31765-027). This medium is furthersupplemented with heat-inactivated 10% fetal calf serum (FCS, JRHBiosciences, Kansas Cat. #12107-1000M), 1% NEAA (MEM Non essential AminoAcid solution (Sigma-Aldrich Cat.# M1745), and 1% Penicillin (10.000U/ml)/Streptomycin (10.000 μg/ml) (Invitrogen Life Technologies, Cat.#15140-122). To stimulate neuronal differentiation and growth ofneuronal processes, SH-SY5Y cells were cultured in medium supplementedwith 10 μM retinoic acid (RA, Sigma-Aldrich Cat. # R2625-050MG)) forseveral days. Differentiated SH-SY5Y cells were grown in tissue cultureflasks and were removed by careful trypsination and were plated on glasscoverslips coated with a striped pattern of RGM A protein or fragment ofit and Collagen I.

(viii) Preparation of striped glass coverslips

The modified version of the stripe assay on glass coverslips wasperformed in a slightly different way as described previously (Knoell etal. Nature Protocols 2: 1216-1224, 2007) and is summarized below.

Sterile silicon matrices for production of stripes consisting ofpurified proteins were pressed on the surface of a petri dish with therough face of the matrix pointing upwards. Ethanol washed, cleancoverslips were laid down onto the matrix and the corners of the matrixare marked with an ink ball point pen at the backside of the coverslip.The matrix carrying the coverslip was carefully turned upside down withthe coverslip facing the bottom of the petri dish. Fc-conjugated fulllength inhibitory RGM A or fc-fragments or recombinant human RGM A (R&DSystems Cat. #2459 RM) of it were mixed with 10 μl of an FITC-labeledanti-mouse antibody (Fab-specific goat anti-mouse IgG, Sigma-AldrichCat. # F-4018) to visualize the RGM A stripes. Using a Hamilton syringe,50 μl of the RGM A-FITC antibody solution is carefully injected throughthe inlet channel. Excess fluid left the matrix through the outletchannel and is removed with a Kleenex cloth. After incubation of thematrix-coverslip at 37° C. for 2 hours, the first coating solution(containing RGM A) was washed away with 100 μl of PBS. In the next step,the coverslip with the RGM A stripes was transferred to a 24 well plate,coated with 500 μl Collagen I (rat tail Collagen I, Becton DickinsonBiosciences Cat. #354236) to fill the empty spaces between the RGM Astripes and was incubated at 37° C. for 2 hours. In the end a pattern ofalternating stripes of RGM A and Collagen I was produced on thecoverslip. After incubation, non-bound Collagen I was washed away bythree separate wash steps with PBS and differentiated SH-SY5Y cells wereplated onto the coverslips. Incubation of the SH-SY5Y cells on thepatterned substrate was continued at 37° C. for 20-24 hours in thepresence or absence of monoclonal antibodies directed against human RGMA.

For immunofluorescence analysis cells were fixed in 4% paraformaldehydefor 2 h at room temperature or overnight at 4° C. and permeabilized byincubation with PBS containing 0.1% Triton X-100 for 10-20 min at roomtemperature. After blocking with 3% BSA for 60 minutes, cells wereincubated with the primary antibody (monoclonal anti-β-tubulin isotype 3clone SDL 3D10, Sigma-Aldrich Cat. # T8660) for 2 hours at roomtemperature and after several wash steps with the secondary antibody(Cy-3 donkey anti-mouse JacksonImmuno Research Lot:62597), diluted inPBS with 0.1% BSA for 1 h. Nuclei were counterstained using bisbenzimideH33258 (Riedel-De-Haen, Cat. # A-0207). Cells were finally embedded inFluoromount G (Southern Biotechnology Associates Inc.: Cat. #010001).Cells were analyzed using an Axioplan2 fluorescence microscope (Zeiss).

(ix) Construction and expression of recombinant anti RGMA antibodies

The DNA encoding the cDNA fragments of the heavy chain variable regionof rat anti-human RGMA monoclonal antibodies 5F9 and 8D1 was cloned intoa pHybE expression vector containing the human IgG1 constant region,which contains 2 hinge-region amino acid mutations, by homologousrecombination in bacteria. These mutations are a leucine to alaninechange at positions 234 and 235 (EU numbering, Lund et al., 1991, J.Immunol., 147: 2657). The light chain variable region of the 5F9 and 8D1monoclonal antibodies were cloned into pHybE vector containing a humankappa constant region. Exemplary pHyb-E vectors include the pHybE-hCk,and pHybE-hCg1,z,non-a (see U.S. Patent Application Ser. No.61/021,282). Full-length antibodies were transiently expressed in 293Ecells by co-transfection of chimeric heavy and light chain cDNAs ligatedinto the pHybE expression plasmid. Cell supernatants containingrecombinant antibody were purified by Protein A Sepharose chromatographyand bound antibody was eluted by addition of acid buffer. Antibodieswere neutralized and dialyzed into PBS. The purified anti-human RGMAmonoclonal antibodies were then tested for their ability to bind RGMA byELISA as described in Example 1 and competition ELISA as described inExample 7.

Example 1 Generation of Anti Human RGMA Monoclonal Antibodies

Anti human RGMA rat monoclonal antibodies were obtained as follows:

Example 1A Immunization of Rats with Human RGMA Antigen

Twenty-five micrograms of recombinant purified human RGMA (R&D SystemsCat#2459-RM lot MRH02511A) mixed with complete Freund's adjuvant(Sigma,) was injected subcutaneously into four 6-8 week-old HarlanSprague Dawley rats on day 1. On days 21, 42, and 63, twenty-fivemicrograms of recombinant purified human RGMA mixed with IncompleteFreunds adjuvant (Sigma) was injected subcutaneously into the same 4Harlan Sprague Dawley rats. On day 144, or day 165 rats were injectedintravenously with 10 μg recombinant purified human RGMA

Example 1B Generation of Hybridoma

Splenocytes obtained from the immunized rats described in Example 1.2.Awere fused with SP2/O-cells at a ratio of 2:1 according to theestablished method described in Kohler, G. and Milstein 1975, Nature,256: 495 to generate hybridomas. Fusion products were plated inselection media containing azaserine and hypoxanthine in 96-well platesat a density of 1.5×10⁵ spleen cells per well. Seven to ten days postfusion, macroscopic hybridoma colonies were observed. Supernatant fromeach well containing hybridoma colonies was tested by direct ELISA (seeExample 2) for the presence of antibody to human RGMA. ELISA positivecell lines were tested in FACS against stable transfected HEK293 cellsexpressing human and/or rat RGMA. These rat hybridoma cell lines weresubsequently tested in Direct ELISA for crossreactivity with rat RGMA,and ELISA binding to HuRGMA 47-168 fusion protein.

TABLE 7 Binding of anti RGMA Rat Monoclonal Antibodies Direct FACSDirect Direct ELISA ELISA HEK293- ELISA hRGMA Name rHuRGMA rhRGMArRatRGMA 47-168/HuIgGFc ML68-8D1 Yes Yes No Yes ML69-5F9 Yes Yes Yes Yes

Example 2 Direct ELISA Binding of mABs 5F9 and 8D1

As shown in FIG. 1A, MABs 5F9 and 8D1 bind to hRGM A with similartiters, as described in above section (i). MAB 5F9 was also shown tobind to ratRGM A in ELISA, while 8D1 is not capable of binding to ratRGMA (data not shown). FIG. 1B shows that MABs 5F9 and 8D1 bind to HEK293cells overexpressing hRGM A in FACS. FIG. 1C shows that 5F9 but not 8D1is capable of binding BAF3 cells overexpressing ratRGM A in FACS. FACSwas carried out as described in section (ii).

Solid phase ELISA assays were used to evaluate MAB 5F9 binding incompetitive hRGM A-neogenin binging assays. ELISA plates were preparedand used as described in section (iii) of the present application. hRGMA was added at a concentration of 0.5 μg/ml with 5F9 antibodies for 1 hat 37° C. MAB 5F9 was used at the following concentrations: 1.25 μg/ml;0.63 μg/ml; 0.32 μg/ml; 0.16 μg/ml; 0.08 μg/ml; 0.04 μg/ml; 0.02 μg/ml;0.01 μg/ml. Binding of hRGM A was visualized using a Biotin-labeledanti-fc antibody and a Streptavidin-Peroxidase complex. Plates wereanalyzed (OD determination) at a wave length of 450 nm using an Anthosphotometer. As shown in FIG. 2, the three highest antibodyconcentrations, dose-dependently inhibited binding of full length humanRGM A to Neogenin.

Solid phase ELISA assays were used also for evaluating MAB 5F9 incompetitive hRGM A-BMP-4 binding assays. ELISA plates were prepared andused as described in section (iv) of the present application. hRGM A wasadded at a concentration of 0.5 μg/ml with 5F9 antibodies for 1 h at 37°C. MAB 5F9 was used at the following concentrations: 1.25 μg/ml; 0.63μg/ml; 0.32 μg/ml; 0.16 μg/ml; 0.08 μg/ml; 0.04 μg/ml; 0.02 μg/ml; 0.01μg/ml. Binding of hRGM A was visualized using a Biotin-labeled anti-fcantibody and a Streptavidin-Peroxidase complex. Plates were analyzed (ODdetermination) at a wave length of 450 nm using an Anthos photometer. Asshown in FIG. 3, the four highest antibody concentrations,dose-dependently inhibited binding of full length human RGM A to BMP-4.

Solid phase ELISA assays were also used for evaluating MAB 5F9 bindinginhibition of fragment 0 (47-168) hRGM A to BMP-4. ELISA plates werecoated for 1 h at 37° C. with a concentration of 2.5 μg/ml of the humanrecombinant BMP-4 protein. hRGM A light chain (fragment 0, 47-168) wasadded at a concentration of 0.5 μg/ml with 5F9 antibodies for 1 h at 37°C. MAB 5F9 was used at the following concentrations: 1.25 μg/ml; 0.63μg/ml; 0.32 μg/ml; 0.16 μg/ml; 0.08 μg/ml; 0.04 μg/ml; 0.02 μg/ml; 0.01μg/ml. Binding of hRGM A was visualized using a Biotin-labeled anti-fcantibody and a Streptavidin-Peroxidase complex. Plates were analyzed (ODdetermination) at a wave length of 450 nm using an Anthos photometer.FIG. 4 depicts the antibody concentrations of 1.25 μg/ml, 0.63 μg/ml and0.32 μg/ml dose-dependently inhibiting binding of the human RGM A lightchain to BMP-4.

Solid phase ELISA assays were also used for evaluating MAB 5F9 bindingin competitive hRGM A-BMP-2 binding assays. ELISA plates were preparedand used as described in section (v) of the present application. Fulllength hRGM A was added at a concentration of 0.5 μg/ml with 5F9antibodies for 1 h at 37° C. MAB 5F9 was used at the followingconcentrations: 5 μg/ml; 2.5 μg/ml; 1.25 μg/ml; 0.63 μg/ml; 0.32 μg/ml;0.16 μg/ml. Binding of hRGM A was visualized using a Biotin-labeledanti-fc antibody and a Streptavidin-Peroxidase complex. Plates wereanalyzed (OD determination) at a wave length of 450 nm using an Anthosphotometer. FIG. 5 depicts antibody concentrations 5 μg/ml, 2.5 μg/ml,1.25 μg/ml, 0.63 μg/ml, inhibiting the binding of full length human RGMA to BMP-2.

Solid phase ELISA assays were also used to evaluate MABs 5F9 and 8D1 inhRGM A-Neogenin, hRGM A-BMP-2 and hRGM A-BMP-4 binding assays. (FIG. 9)As described, ELISA plates were coated for 1 h at 37° C. with aconcentration of 2.5 μg/ml of the extracellular domain of the His-taggedhuman Neogenin protein or with 2.5 μg/ml Bmp-2 or BMP-4. Full lengthfc-conjugated hRGM A was added at a concentration of 0.5 μg/ml withantibodies for 1 h at 37° C. MABs 5F9 and 8D1 were used at the followingconcentrations: 5 μg/ml; 2.5 μg/ml; 1.25 μg/ml; 0.63 μg/ml; 0.32 μg/ml;0.16 μg/ml; 0.08 μg/ml. Binding of hRGM A was visualized using aBiotin-labeled anti-fc antibody and a Streptavidin-Peroxidase complex.Plates were analyzed (OD determination) at a wave length of 450 nm usingan Anthos photometer. As shown in FIG. 9, the rat monoclonal antibody8D1 inhibits or reduces binding of human RGM A to BMP-2 and to BMP-4 butis not able to inhibit its binding to Neogenin.

Example 3 mAb 5F9 Activity in Neurite Growth Assays with Aggregates ofDifferentiated Human Ntera Neurons

Ntera cells were obtained and cultured as described in Method section(vi) of the present application. mAb 5F9 neutralized the neuriteoutgrowth inhibitory activity of the potent fc-conjugated light chain(amino acids 47-168) of the human RGM A protein in neurite growth assayswith aggregates of differentiated human NTera neurons. As shown in FIG.6, in the absence of an inhibitory RGM A protein or fragment and in thepresence of the outgrowth-stimulating substrate laminin, neuronal NTeraaggregates show an extensive and dense network of outgrowing neurites(A). Also shown in FIG. 6, the presence of the hRGM A light chain,dramatically reduces number, density and length of NTera neurites,proving the potent inhibitory activity of the hRGM A fragment. The fewneurites leaving the aggregate are short and tightly bundled (B). PartsC-E of FIG. 6 show increasing concentrations of the MAB 5F9, added tothe cultures does-dependently neutralized or derepressed theneurite-growth inhibitory activity of the hRGM A light chain fragment.With increasing MAB concentrations, outgrowth of NTera neuronalaggregates is completely restored, despite the presence of the RGM Ainhibitor (C: 0.1 μg/ml MAB 5F9; D: 1 μg/ml MAB 5F9; E: 10 μg/ml MAB5F9).

Quantitative analysis of the neutralising activity of MAB 5F9 in neuritegrowth assays with human NTera aggregates was performed to test thepotent fc-conjugated light chain inhibitory fragment (amino acids47-168) of the human RGM A protein. Outgrowth of the cultures wasautomatically analyzed by having aggregates stained with bisbenzimideand subsequently photographed. The staining only marked the aggregatenot the outgrowing neurites. These were however stained with an antibodyto B3-tubulin and a fluorophor-labeled secondary antibody. Neuriteoutgrowth was automatically determined by calculating the neuriteoutgrowth index, an index determined by subtracting the area of the cellbodies from the β3-tubulin stained area of the aggregate and itsprocesses. This factor was then divided by the area of the cell bodiesas described in Lingor et al. J. Neurochem. 103: 181-189, 2007. FIG. 7,shows that MAB 5F9 dose-dependently (0.1-10.0 μg) neutralized theoutgrowth inhibitory activity of an fc-conjugated, potent hRGM Ainhibitor fragment (fragment 0, 47-168; 10 μg) in neurite growth assayswith human Ntera aggregates.

Example 4 mAb 5F9 Activity in hRGM A/Collagen I Stripes

SH-SY5Y cells were cultured and used as described in section (vii) ofthe present application. Striped glass coverslips with RGM A andCollagen I were prepared as described in section (viii) of the presentapplication. A carpet with alternating stripes of hRGM A/Collagen I andCollagen I was produced for the experiments according to a protocoldescribed in the literature (Knoell et al. Nature Protocols 2:1216-1224, 2007). In the absence of the 5F9 MAB (A), neuronal SH-SY5Ycells show a clear preference for the Collagen I stripe with more than90% of the cells preferring Collagen I stripes over hRGM A stripes. Withincreasing concentrations of MAB 5F9 neuronal SH-SY5Y cells prefer hRGMA stripes over Collagen I stripes (B-E). At the highest MABconcentration used (E)), SH-SY5Y neurons show a strong preference forhRGM A stripes in comparison with the Collagen I stripes (see FIG. 8).This can be interpreted as a unique characteristic of the 5F9 MAB sinceit transformed the inhibitory nature of RGM A in an attractive activity.In the presence of increasing concentrations of 5F9, neuronal cellsprefer to migrate and grow on an RGM A substrate, and not on apermissive substrate like Collagen I. Such a unique feature has neverbeen described before for a monoclonal antibody.

Example 5 Construction of CDR-Grafted Antibodies

By applying standard methods well known in the art, the CDR sequences ofVH and VL chains of monoclonal antibody 5F9 (see Table 5 above) aregrafted into different human heavy and light chain acceptor sequences.Based on sequence VH and VL alignments with the VH and VL sequences ofmonoclonal antibody 5F9 of the present invention the following knownhuman sequences are selected:

-   -   a) VH3-48, VH3-33 and VH3-23 as well as the joining sequences        hJH3, hJH4 and hJH6 for constructing heavy chain acceptor        sequences (according to Table 3 above);    -   b) A17 and A18 as well as hJK2 for constructing light chain        acceptor sequences (according to Table 4 above).        By grafting the corresponding VH and VL CDRs of 5F9 into said        acceptor sequences the following CDR grafted, humanized,        modified VH and VL sequences were prepared (see also Table 6,        above): VH 5F9.1-GL, VH 5F9.2-GL, VH 5F9.3-GL, VH 5F9.4-GL, VH        5F9.5-GL, VH 5F9.6-GL, VH 5F9.7-GL, and VH 5F9.8-GL; VL        5F9.1-GL, VL 5F9.2-GL, and VL 5F9.3-GL.

Example 6 Construction of Framework Back Mutations in CDR-GraftedAntibodies

To generate humanized antibody framework back mutations, mutations areintroduced into the CDR-grafted antibody sequences as prepared accordingto Example 5, by de novo synthesis of the variable domain and/or usingmutagenic primers and PCR, and methods well known in the art. Differentcombinations of back mutations and other mutations are constructed foreach of the CDR-grafts as follows.

For heavy chains VH 5F9.1-GL, VH 5F9.2-GL, and VH 5F9.3-GL one or moreof the following Vernier and VH/VL interfacing residues are back mutatedas follows: V37→I, V48→I, S49→G, and/or R98→K

For heavy chains VH 5F9.4-GL, VH 5F9.5-GL, and VH 5F9.6-GL one or moreof the following Vernier and VH/VL interfacing residues are back mutatedas follows: V37→I, V48→I, A49→G, R98→K.

For heavy chains VH 5F9.7-GL, VH 5F9.8-GL, and VH 5F9.9-GL one or moreof the following Vernier and VH/VL interfacing residues are back mutatedas follows: V37→I, V48→, S49→G.

Additional mutations include the following:

-   -   for heavy chains VH 5F9.1-GL, VH 5F9.2-GL, and VH 5F9.3-GL:        D88→A,    -   for heavy chains VH 5F9.4-GL, VH 5F9.5-GL, and VH 5F9.6-GL: L1→E        and    -   for heavy chains VH 5F9.7-GL, VH 5F9.8-GL, and VH 5F9.9-GL:        L5→V.    -   For light chain VL 5F9.1-GL one or more of the following Vernier        and VH/VL interfacing residues are back mutated as follows:        I2→V, M4→L, Y41→F.    -   For light chain VL 5F9.2-GL one or more of the following Vernier        and VH/VL interfacing residues are back mutated as follows:        M4→L, R51→L.    -   For light chain VL 5F9.3-GL one or more of the following Vernier        and VH/VL interfacing residues are back mutated as follows:        M4→L, Y41→F.

Example 7 Construction and Expression of Recombinant Humanized Anti RGMAAntibodies

pHybE expression vectors harboring heavy and light chains containingframework back mutations were co-transfected into 293-6E cells totransiently produce full-length humanized antibodies as described insection ix above. Mutations were introduced into the CDR-graftedantibody sequences as prepared according to Example 5, by de novosynthesis of the variable domain and/or using mutagenic primers and PCR,and methods well known in the art. The amino acid sequences of the VHand VL regions of the humanized antibodies are disclosed in Table 8.

Specifically, for the heavy chains:

VH 5F9.1, VH 5F9.5, and VH 5F9.9 contain VH 5F9.4-GL with a Q1→Emutation.

VH 5F9.2, VH 5F9.6, VH 5F9.10, VH 5F9.19, VH 5F9.20, VH 5F9.21, and VH5F9.22 contain VH 5F9.4-GL with a Q1→E mutation and the followingVernier and VH/VL interfacing residue back mutations: V37→I, V48→I,A49→G, R98→K.

VH 5F9.3, VH 5F9.7, and VH 5F9.11 contain VH 5F9.7-GL with a L5→Vmutation. VH 5F9.4, VH 5F9.8, VH 5F9.12, VH 5F9.23, VH 5F9.24, VH5F9.25, and VH 5F9.26 contain VH 5F9.7-GL with a L5→V mutation and thefollowing Vernier and VH/VL interfacing residue back mutations: V37→I,V48→I, S49→G.

For the light chains:

VL 5F9.1, VL 5F9.2, VL 5F9.3, and VL 5F9.4 are identical to VL 5F9.1-GL.

VL 5F9.5, VL 5F9.6, VL 5F9.7, and VL 5F9.8 are identical to VL 5F9.2-GL.

VL 5F9.9, VL 5F9.10, VL 5F9.11, and VL 5F9.12 are identical to VL5F9.3-GL.

VL 5F9.19 and VL 5F9.23 contain VL 5F9.2-GL with the following Vernierand VH/VL interfacing residue back mutations: M4→L, R51→L. VL 5F9.20 andVL 5F9.24 contain VL 5F9.2-GL with the following Vernier and VH/VLinterfacing residue back mutation: M4→L.

VL 5F9.21 and VL 5F9.25 contain VL 5F9.3-GL with the following Vernierand VH/VL interfacing residue back mutations: M4→L, Y41→F. VL 5F9.22 andVL 5F9.26 contain VL 5F9.3-GL with the following Vernier and VH/VLinterfacing residue back mutation: M4→L.

TABLE 8 Expression of humanized antibodies SEQ ID Protein Sequence No.region 123456789012345678901234567890 47 VH h5F9.1EVQLVESGGGVVQPGRSLRLSCAASGFTFS NYGMNWVRQAPGKGLEWVAMIYYDSSEKHYADSVKGRFTISRDNAKNTLYLQMNSLRAED TAVYYCARGTTPDYWGQGTMVTVSS 44 VL h5F9.1DIVMTQTPLSLSVTPGQPASISVRSSQSLE YSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 48 VH h5F9.2EVQLVESGGGVVQPGRSLRLSCAASGFTFS NYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 44 VL h5F9.2DIVMTQTPLSLSVTPGQPASISCRSSQSLE YSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 49 VH h5F9.3EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYGMNWVRQAPGKGLEWVSMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 44 VL h5F9.3DIVMTQTPLSLSVTPGQPASISCRSSQSLE YDDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 50 VH h5F9.4EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 44 VL h5F9.4DIVMTQTPLSLSVTPGQPASISCRSSQSLE YSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHPLTFGQGTKLEIKR 47 VH h5F9.5EVQLVESGGGVVQPGRSLRLSCAASGFTFS NYGMNWVRQAPGKGLEWVAMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARGTTPDYWGQGTMVTVSS 45 VL h5F9.5DVVMTQSPLSLPVTLGQPASISCRSSQSLE YSDGYTFLEWFQQRPGQSPRRLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 48 VH h5F9.6EVQLVESGGGVVQPGRSLRLSCAASGFTFS NYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 45 VL h5F9.6DVVMTQSPLSLPVTLGQPASISCRSSQSLE YSDGYTFLEWFQQRPGQSPRRLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 49 VH h5F9.7EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYGMNWVRQAPGKGLEWVSMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 45 VL h5F9.7DVVMTQSPLSLPVTLGQPASISCRSSQSLE YSDGYTFLEWFQQRPGQSPRRLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 50 VH h5F9.8EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 45 VL h5F9.8DVVMTQSPLSLPVTLGQPASISVRSSQSLE YSDGYTFLEWFQQRPGQSPRRLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 47 VH h5F9.9EVQLVESGGGVVQPGRSLRLSCAASGFTFS NYGMNWVRQAPGKGLEWVAMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARGTTPDYWGQGTMVTVSS 46 VL h5F9.9DVVMTQSPLSLPVTLGQPASISVRSSQSLE YSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 48 VH h5F9.10EVQLVESGGGVVQPGRSLRLSCAASGFTFS NYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 46 VL h5F9.10DVVMTQSPLSLPVTLGQPASISVRSSQSLE YSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 49 VH h5F9.11EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYGMNWVRQAPGKGLEWVSMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 46 VL h5F9.11DVVMTQSPLSLPVTLGQPASISCRSSQSLE YSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 50 VH h5F9.12EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 46 VL h5F9.12DVVMTQSPLSLPVTLGQPASISCRSSQSLE YSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 48 VH h5F9.19EVQLVESGGGVVQPGRSLRLSCAASGFTFS NYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 51 VL h5F9.19DVVLTQSPLSLPVTLGQPASISCRSSQSLE YSDGYTFLEWFQQRPGQSPRLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 48 VH h5F9.20EVQLVESGGGVVQPGRSLRLSCAASGFTFS NYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 52 VL h5F9.20DVVLTQSPLSLPVTLGQPASISCRSSQSLE YSDGYTFLEWFQQRPGQSPRLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 48 VH h5F9.21EVQLVESGGGVVQPGRSLRLSCAASGFTFS NYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 53 VL h5F9.21DVVLTQSPLSLPVTLGQPASISCRSSQSLE YSDGYTFLEWFLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 48 VH h5F9.22EVQLVESGGGVVQPGRSLRLSCAASGFTFS NYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 54 VL h5F9.22DVVLTQSPLSLPVTLGQPASISCRSSQSLE YSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 50 VH h5F9.23EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 51 VL h5F9.23DVVLTQSPLSLPVTLGQPASISCRSSQSLE YSDGYTFLEWFQQRPGQSPRLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 50 VH h5F9.24EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 52 VL h5F9.24DVVLTQSPLSLPVTLGQPASISCRSSQSLE YSDGYTFLEWFQQRPGQSPRLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 50 VH h5F9.25EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 53 VL h5F9.25DVVLTQSPLSLPVTLGQPASISCRSSQSLE YSDGYTFLEWFLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR 50 VH h5F9.26EVQLVESGGGLVQPGGSLRLSCAASGFTFS NYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGTTPDYWGQGTMVTVSS 54 VL h5F9.26DVVLTQSPLSLPVTLGQPASISCRSSQSLE YSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQATHDPLTFGQGTKLEIKR

Example 8 Characterization of Humanized 5F9 Antibodies Using CompetitionELISA

ELISA plates (Costar 3369) were coated overnight at 4° C. with 50μL1/well of 0.25 μg/ml hRGMA in 0.2 M sodium carbonate-bicarbonatebuffer, pH 9.4, washed with Wash Buffer (PBS containing 0.1% Tween 20),and blocked for 1 hr at room temperature with 200 μl/well of 2% nonfatdry milk in PBS. After washing with Wash Buffer, a mixture of abiotinylated chimeric 5F9 (0.1 μg/ml final concentration) and unlabelledcompetitor test antibody starting at 50 μg/ml final concentration andserially diluted 5-fold) in 50 μL1/well of ELISA buffer was added induplicate. After incubating the plates for 1 hr at room temperature, andwashing with Wash Buffer, bound antibodies were detected using 100μl/well of 1:10,000 dilution of HRP-conjugated streptavidin (Fitzgerald)in ELISA buffer. After incubating for 1 hr at room temperature, andwashing with Wash Buffer, color development was performed by adding 100μl/well of TMB Buffer (Zymed). After incubating for 15 min at roomtemperature, color development was stopped by adding 50 μL1/well of 1Nhydrochloric acid. Absorbance was read at 490 nm.

Table 9 shows the IC₅₀ values of humanized 5F9 antibodies obtained usingthe computer software GraphPad Prism (GraphPad Software Inc., San Diego,Calif.).

TABLE 9 IC50 values of humanized 5F9 antibodies in competitive ELISAIC50 Antibody (μg/ml) h5F9.1 >10 h5F9.2 >10 h5F9.3 >10 h5F9.4 >10h5F9.5 >10 h5F9.6 >10 h5F9.7 >10 h5F9.8 >10 h5F9.9 >10 h5F9.10 >10h5F9.11 >10 h5F9.12 >10 h5F9.19 N/A h5F9.20 >2.0 h5F9.21 0.60h5F9.22 >2.0 h5F9.23 0.55 h5F9.24 1.32 h5F9.25 0.66 h5F9.26 >2.0

Example 9 Affinity Determinations of Chimeric and Humanized AntibodiesUsing BIACORE technology

The BIACORE assay (Biacore, Inc, Piscataway, N.J.) determines theaffinity of antibodies with kinetic measurements of on-, off-rateconstants. Binding of antibodies to recombinant purified human RGMA wasdetermined by surface plasmon resonance-based measurements with aBiacore® 3000 instrument (Biacore® AB, Uppsala, Sweden) using runningHBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005%surfactant P20) at 25° C. All chemicals were obtained from Biacore® AB(Uppsala, Sweden). Approximately 5000 RU of goat anti-human IgG, (Fcγ),fragment specific polyclonal antibody (Pierce Biotechnology Inc,Rockford, Ill.) diluted in 10 mM sodium acetate (pH 4.5) was directlyimmobilized across a CM5 research grade biosensor chip using a standardamine coupling kit according to manufacturer's instructions andprocedures at 25 μg/ml. Unreacted moieties on the biosensor surface wereblocked with ethanolamine. Modified carboxymethyl dextran surface inflowcell 2 and 4 was used as a reaction surface. Unmodifiedcarboxymethyl dextran without goat anti-human IgG in flow cell 1 and 3was used as the reference surface. Purified antibodies were diluted inHEPES-buffered saline for capture across goat anti-human IgG specificreaction surfaces. Human antibodies to be captured as a ligand (25μg/ml) were injected over reaction matrices at a flow rate of 5 μl/min.The association and dissociation rate constants, k_(on) (unit M⁻¹ s⁻¹)and k_(off) (unit s⁻¹) were determined under a continuous flow rate of25 μl/min. Rate constants were derived by making kinetic bindingmeasurements at ten different antigen concentrations ranging from0.39-50 nM. For kinetic analysis, rate equations derived from the 1:1Langmuir binding model were fitted simultaneously to association anddissociation phases of all eight injections (using global fit analysis)with the use of Bioevaluation 4.0.1 software. The equilibriumdissociation constant (unit M) of the reaction between humanizedantibodies and recombinant purified human RGMA was then calculated fromthe kinetic rate constants by the following formula:K_(D)=k_(off)/k_(on).

TABLE 9 Affinity of chimeric and humanized anti-RGMA MonoclonalAntibodies Name k_(on) (1/M · s) k_(off) (1/s) K_(D) (nM) chimeric 5F97.65 × 10⁵ 2.36 × 10⁻³ 3.09 h5F9.21 3.55 × 10⁵ 2.69 × 10⁻³ 7.59 h5F9.235.07 × 10⁵ 2.21 × 10⁻³ 4.37 h5F9.25 5.70 × 10⁵ 3.29 × 10⁻³ 5.78

Example 10 The Humanised 5F9 Antibodies Neutralise ChemorepulsiveActivity of Human RGM A in a Neuronal SH-SY5Y Chemotaxis Assay

The chemotaxis assay measures chemotactic behaviour of cells in responseto diffusible factors which can exert chemoattractive or chemorepulsiveactivities. RGM A has been described as a protein acting in bothmembrane-bound (contact-dependent repulsion) and in soluble, diffusibleform (chemorepulsive) and has therefore been evaluated in an hRGM Achemotaxis assay. To this aim RGM A-sensitive human neuroblastoma cellsSH-SY5Y, carrying the RGM receptor Neogenin were used (Schaffar et al.J. Neurochemistry: 107: 418-431, 2008). SH-SY5Y cells were grown inEarle's Balanced Salt Solution/F12 (EBSS/F12) medium supplemented with10% fetal bovine serum and 1% non-essential amino acids (MEM-NEAA). Forinduction of neurite outgrowth cells were cultured in mediumsupplemented with 10 μM retinoic acid (RA). 5-6 hours later, cells weretrypsinized and counted for plating in 24-well Boyden Chambers (BDFalcon 351185, HTS Multiwell System). 500 μl of the cell suspension(corresponding to 1×10⁵ cells) was added to the inner circle of eachwell. This inner circle is separated from the larger outer circle ofeach well by a PET membrane with 8 μm pore diameter. 600 μl of medium+/−RGM A +/− antibodies were pipetted into the outer circle and cellswere cultivated in the Multiwell Boyden Chambers overnight at 37° C.After incubation medium was aspirated and replaced by the fixative (2%paraformaledhyde. Fixation was continued for 2 hours at room temperatureand after several wash steps with PBS permeabilization was performedusing PBS containing 0.1% Triton-X-100 (15 min, RT). Staining of cellswas done by incubating them for 1 hour in the dark in a solution ofAlexa Fluor 488 Phalloidin 1:100 (Invitrogen A12379) and Bisbenzimide(H332456) 1:100. After 2 wash steps with PBS, cultures were filled byPBS, sealed by parafilm and stored in the dark for the analysis with afluorescence microscope (Zeiss Axiovert).

In the absence of hRGM A cells migrate through the membrane pores andcan be counted after fixation and staining. Only those cells are countedwhich attached to the bottom of the membrane, because these cells hadmigrated through the PET membrane. Cells on the upper side of themembrane were carefully removed before the fixation procedure. Thischemotaxis assay proved that presence of hRGM A significantly reducedthe number of SH-SY5Y cells migrating through the membrane by more than80%. The rat monoclonal 5F9, the chimeric human-rat 5F9 and thehumanised 5F9 but not an isotype control rat monoclonal antibody (p21)partially or completely neutralised chemorepulsive activity of hRGM A at10 μg/ml, manifested as larger numbers of cells found at the bottom ofthe membrane (FIG. 10).

Example 11 5F9 Induces Regeneration of Crushed, Damaged Myelinated OpticNerve Axons in a Rat Model of Optic Nerve Injury

The model of Optic Nerve Crush (or Optic Nerve Injury) provides ananimal model to test various substances that stimulate regeneration ofthe optic nerve fibers and reduce the massive cell death of retinalganglion cells.

The experiments were carried out in adult male Sprague Dawley and maleWistar rats obtained from Charles River (D) Laboratories (Germany). Theanimals are kept in single cages on a 12:12 h light/dark cycle with foodand water ad libitum. The optic nerve crush is performed always only atthe left eye by minimal anterior surgery. This is a minimally invasivemethod of optic nerve injury and was developed by us, according to humananterior visual surgical methods. Before and during the operationprocedure animals are anesthetized by inhalation anesthesia usingSevoflurane (Abbott GmbH Co. & KG, Delkenheim, Germany) and are fixed onthe operation table by using jawclamp and adhesive tape for the limbs. Adrop in body temperature is prevented by mounting animals on a heatingpad. For anterior crush surgery of the rat optic nerve, the left eye iscarefully freed of ligament and connective tissue. As a first step, amicrosurgical cut (2-3 mm) of the adjacent tissue in the outer corner ofthe eye is performed. Then the optic nerve is exposed by using a pair offorceps to move to the side the eye muscles and lacrimal gland, thussparing it. At the next step, the meninges were longitudinally opened byusing microscissors to expose the optic nerve.

This results in a higher mobility of the eye and enables lateralrotation of the eye and access to its left optic nerve. The optic nerveis injured approximately 1-3 mm behind the eye, using a pair of forcepsset to provide a fixed maximum pressure for 20-30 s. Special care istaken not to damage the vascular supply to the eye.

a) Local Administration of Antibodies and Buffer Solution.

After crush injury of the optic nerve male Sprague Dawley rats weretreated locally with 5F9 antibody (n=10 animals), the 8D1 controlantibody (n=10 animals) or with a vehicle control PBS (n=10 animals).Experimenters were blinded for the different treatment groups. For localantibody application, small gelfoam pieces (length: 2.5 mm, width: 2.5mm, height: 2.5 mm) were soaked with 20 μl of a 10 mg/ml antibodysolution or with 20 μl of PBS and were placed directly adjacent to theoptic nerve lesion site. After minimal invasive surgery and antibodyapplication, animals were placed on paper towels in the clean cagemounted on the warmer to control the body temperature until they startedto move. An ointment which contains antibiotic (Gentamytrex, Dr. MannPharma) was applied onto the eye to avoid bacterial infection anddrying-out of the sclera. Carprofen (Rimadyl, 5 mg/kg, Pfizer GmbH,Karlsruhe) was applied i.p. for postoperative pain therapy directlyafter surgery and then twice per day for a 3 days period. The animalswere observed and controlled regularly several hours directly aftersurgery and in the next days to make sure that all the animals survivedand recovered from anesthesia and surgery. 5 weeks after surgery andantibody/vehicle application, animals were anesthetized with an overdoseof Narcoren (40-60 mg/kg) and were perfused by injection of 4%paraformaledyde solution into the heart. Optic nerves were isolated andwere transferred into a 4% paraformaldeyde solution for 1 h at roomtemperature to ensure proper fixation of the tissue. Followingpostfixation, rat optic nerves were stored over night in a 30% sucrosesolution (4° C.). On the following day optic nerves were embedded inTissue Tek, frozen and longitudinal sections with a thickness of 16 μmwere made using a Cryostat.

For immunostainings, optic nerve sections were fixed with cold (−20° C.)Acetone (10 min), washed 3× (5 min) with Tris Buffered Saline (TBS,Fluka 93312) and were blocked and permeabilised with TBS, containing 5%Bovine Serum Albumin and 1% Triton-X-100 (30 min), at room temperature).Residual BSA and detergent was removed by 2 separate wash steps (5 mineach) with TBS. Sections were incubated for 1 h at room temperature witha polyclonal rabbit anti-GAP-43 antibody (Abcam, ab 7562) diluted 1:100in 5% BSA/TBS solution. After 3 wash steps with TBS, 0.1% Tween,sections were incubated for 1 h at room temperature with an Alexa Fluor488-conjugated goat anti-rabbit secondary antibody (Molecular ProbesA11034), diluted 1:1000 in 5% BSA/TBS, containing a 1:100 dilution ofBisbenzimid (H33258, 50 μg/ml) to visualize cell nuclei. Beforeembedding, stained sections were washed 3 times with TBS 0.1% Tween (5min each step) and with distilled water. Sections were embedded inFluoromount G, were covered by a coverslip and were stored in the darkfor microscopic documentation.

Using a Zeiss fluorescence microscope images (FIG. 11) of stainedlongitudinal sections were stored using the Zeiss Axiovison software.Single pictures of each nerve were mounted for analysis using PhotoshopImage Analysis software (Adobe). Quantitative analysis was done in twodifferent ways using the composite images of the optic nerves. TheGAP-43-positive area at the lesion site was measured using theAxiovision software (FIG. 12B). Independent of this first quantitativeanalysis single regenerating fibers (GAP-43 positive) were counted in 4different areas: 0-200 μm, 200-400 μm, 400-600 μm and 600-1200 μm beyondthe crush site. Data analysis and statistical evaluation of data wasdone with the help of the Graphpad Prism software. (FIG. 12A)

b) Systemic Administration of Antibodies and Buffer Solution.

For systemic antibody delivery, male Wistar rats were treatedsystemically (intraperitoneally, ip) or intravenously, iv) with 5F9antibody (n=10 animals) or with a vehicle control PBS (n=10 animals).Animals were injected two times and injections were done on day 0shortly after inducing nerve crush and on day 21 after crush. Doses ofantibody given were 2 mg/kg on day 0 and 10 mg/kg on day 21. Animalswere killed five weeks after crush injury and tissue isolation,preparation of sections, stainings and quantitative analysis was done asdescribed above. As before experimenters were blinded for the twodifferent treatment groups. Composite images of rat optic nerves areshown in FIG. 13. In the 5F9 treated animals (A), many GAP-43 positivefibers are extending beyond the crush site in contrast to controlanimals treated with PBS (B). The crush site is located at the leftmargin and regenerating fibers are stained with an antibody to GAP-43.Many fibers are observed at the upper and lower rim of the optic nervein 5F9-treated animals but not in PBS animals.

5F9 but not the vehicle control PBS significantly increased the numberof regenerating GAP-43 positive fibers. Significantly more fibers(p<0.001) were found in animals treated with 5F9 at distances 300 μm to1800 μm, than in vehicle treated animals. Animals were treated with 5F9at day 0 and d21 with 2 mg/kg and 10 mg/kg, respectively. Antibody orvehicle were given intraperitoneally or intravenously. Data are fromanalysis of 9 animals per group. Per animal 3 series of cryostatsections were analyzed. (FIG. 14A)

In a second experiment, male Wistar rats were treated after optic nerveinjury systemically (iv) with 5F9 antibody (n=10 animals), the 8D1control antibody (n=10 animals) or with the vehicle control PBS (n=10animals). Rats were injected once per week with 2 mg/kg of antibodygiven iv and injections were started immediately after optic nervecrush. All rats received 4 injections and animals were euthanized 5weeks after crush injury. Experimenters were blinded and tissueprocessing and quantitative analysis was done as described before. 5F9but not the vehicle control PBS significantly increased the number ofregenerating GAP-43 positive fibers. Significantly more fibers (p<0.001)were found in animals treated with 5F9 at distances 200 μm to 1400 μm,than in vehicle or control antibody treated animals. Animals weretreated iv once per week for 4 weeks starting at day 0 with 5F9 (2 mg/kgper dose), with the control antibody 8D1 (2 mg/kg per dose) or with PBS.(FIG. 14B)

Example 12 5F9 Induces Remyelination of Crushed, Damaged Optic NerveAxons in a Rat Model of Optic Nerve Injury

A marker for oligodendrocytes and myelin is myelin-basic protein (MBP).An antibody directed against MBP was used to answer the question ifdifferences occurred in remyelination in the different treatment groups.To visualize the process of remyelination, optic nerve sections ofanimals treated systemically were fixed with cold (−20° C.) Acetone (10min), washed 3× (5 min) with Tris Buffered Saline (TBS, Fluka 93312) andwere blocked and permeabilised with TBS, containing 5% Bovine SerumAlbumin and 1% Triton-X-100 (30 min), at room temperature). Residual BSAand detergent was removed by 2 separate wash steps (5 min each) withTBS. Sections were incubated for 3 h or over night at 4° C. with apolyclonal rabbit anti-MBP antibody (Abcam, ab 2404) diluted 1:50 in 5%BSA/TBS solution. After 3 wash steps with TBS, 0.1% Tween, sections wereincubated for 1 h at room temperature with an Alexa Fluor 488-conjugatedgoat anti-rabbit secondary antibody (Molecular Probes A11034), diluted1:1000 in 5% BSA/TBS, containing a 1:100 dilution of Bisbenzimid(H33258, 50 μg/ml) to visualize cell nuclei. Before embedding, stainedsections were washed 3 times with TBS 0.1% Tween (5 min each step) andwith distilled water. Sections were embedded in Fluoromount G, werecovered by a coverslip and were stored in the dark for microscopicdocumentation.

Using a Zeiss fluorescence microscope images of stained longitudinalsections were stored using the Zeiss Axiovison software. Single picturesof each nerve were mounted for analysis using Photoshop Image Analysissoftware (Adobe). Quantitative analysis was done in two different waysusing the composite images of the optic nerves. The MBP-positive area atthe lesion site was measured using the Axiovision software. Dataanalysis and statistical evaluation of data was done with the help ofthe Graphpad Prism software.

Animals were treated with 5F9 at day 0 and d21 with 2 mg/kg and 10mg/kg, respectively. Antibody or vehicle were given intraperitoneally orintravenously. Composite images of rat optic nerves.

Myelination is visualized using an antibody directed against the myelinmarker myelin basic protein MBP. Crush sites are located in the middleof the composite nerves and the area is free in vehicle treated controlanimals (A and B). In the 5F9 treated animals (C and D), manyMBP-positive structures are observed in the middle area (crush center)of the optic nerves. (FIG. 15)

Myelination is visualized using an antibody directed against the myelinmarker myelin basic protein MBP. The MBP area was measured using theZeiss Axiovison software. M1 and M2 are two independent measurements andM is the average measured MPB-positive area. 5F9 increases significantly(p<0.001 versus the vehicle control) the MBP-area of the optic nervecrush site by a factor of 3.5. (FIG. 16)

Example 13 5F9 Protects the Retinal Nerve Fiber Layer (RNLF) fromDegeneration and Induces Sprouting of Retinal Neurons in theDegenerating RNFL

In order to observe protection of RNLF degeneration and induction ofretinal neuron sprouting a novel laboratory assay method wasestablished. Said method is based on explanting and analyzing adult ratretina from the eyes of rats with optic nerve crush (as described inExample 11, above) and systemically treated with antibody 5F9, controlantibody p21 (a rat IgG1 monoclonal antibody) or PBS vehicle. Forperforming this experiment rats with optic nerve crush were treated withAbs or PBS immediately upon inducing the optic nerve crush and then inintervals of 1 week (for five times; at 2 mg/kg or 10 mg/kg dosages ofantibodies)

a) Retina Preparation and Immunofluorescent Staining:

Animals were deep anesthetized with Sevoflurane (8%; Abbott), thenimmediately sacrificed by opening the ribcage and perfusion with 4%paraformaldehyde (PFA) solution through the left heart ventricle. Theeyes were dissected with the connective tissue adjusted and placed in 4%PFA until preparation of retina will take place.

The preparation of the retina was performed in Hank's Balanced SaltSolution (HBSS, Magnesium- and Calcium-free; Invitrogen, #14170070). Theeye was fixed at the connective tissue by tweezers and a round cut wasmade in sclera just around the cornea.

The lens and the ciliary body were carefully removed through theopening, in most cases together with the retina. In case the retina wasstill attached to the sclera—it was gently detached and pulled out.

The half-sphere of retina was cut in four points, opened and spread on agray nitrocellulose membrane (Sartorius, #13006-50-N). If it wasnecessary the membrane with retina on it was allowed to air dry for 5-10sec.

Thereafter, the retina on membrane was placed in 10% neutralphosphate-buffered formaldehyde solution (pH 7.3; Fisher Scientific,#F/1520/21) at +4° C. until the immunofluorescent staining wasperformed.

Staining is performed according to the following protocol:

The retina preparation was washed with TBS, followed by blocking andpermeabilisating with 5% BSA, 1% Triton X-100 in TBS for 30 min andwashing again with TBS. Primary antibodies (monoclonal Ab TUJ-1, a mouseanti-β III Tubulin Ab, AbCam, # ab14545; 1:500 dilution, in TBS, 5% BSA)was added for 1 h at room temperature in the dark followed by washingwith TBS, 0.1% Tween 20. Then secondary antibody (Donkey anti-mouse Cy3;Jackson ImmunoResearch (Dianova) 715-165-151, 1:1000 dilution) andBisbenzimid (50 μg/ml 1:100 dilution) (in TBS, 5% BSA) was added for 1 hat room temperature in the dark followed by washing with TBS, 0.1% Tween20, and washing with desalted H₂O. The preparation was then mounted withFluoromount G and stored at +4° C. in the dark

b) Investigating the Protective Effect of 5F9 on Retinal Fibers in theEye (the RNFL)

Using the Axiovision software, randomly selected images (n=12) of eachretina were chosen and the number of nerve fibers was determined foreach image.

For the experiment three retinae with crushed optic nerves were used foreach group: the 5F9 mab group, the p21 control mab group and the PBSvehicle group. Data analysis and statistical analysis was performedusing the GraphPad prism program.

The results are illustrated in FIG. 17. A significantly higher densityof nerve fiber bundles is observed in retinae of animals systemicallytreated with the 5F9 antibody of the invention.

c) Investigating the Effect of 5F9 on the Sprouting of Retinal Neuronsin the Eye

Using the Axiovision software, randomly selected images (n=12) of eachexplanted retina were chosen and the number of sprouting neurons wasdetermined for each image.

For the experiment three retinae with crushed optic nerves were used foreach group: the 5F9 mab group, the p21 control mab group and the PBSvehicle group. Data analysis and statistical analysis was performedusing the GraphPad prism program.

The results are illustrated in FIG. 18. A significantly higher number ofsprouting intraretinal neurons is observed in retinae of animalssystemically treated with the 5F9 antibody of the invention.

What is claimed is:
 1. A method of treating retinal nerve fiber layer(RNFL) degeneration, comprising the step of administering to a mammal inneed thereof an effective amount of a composition comprising an isolatedantibody, wherein the isolated antibody comprises an antigen bindingdomain, said antibody capable of binding an epitope of a retinalguidance molecule (RGM), said antigen binding domain comprising a heavychain variable domain having three complementary determining regions anda light chain variable domain having three complementary determiningregions, wherein the three complementary determining regions of theheavy chain variable domain have the amino acid sequence of SEQ IDNO:57, SEQ ID NO:58 and SEQ ID NO:59 and the three complementarydetermining regions of the light chain variable domain have the aminoacid sequence of SEQ ID NO:60, SEQ ID NO:61 and SEQ ID NO:62.
 2. Themethod of claim 1, wherein said treatment is an therapeutic,neuroregenerative or neuroprotective, local or systemic treatment. 3.The method of claim 1, wherein the antibody is a monoclonal antibody, achimeric antibody or a humanized antibody.
 4. The method of claim 1,wherein said antibody further comprises a human acceptor framework, andwherein said human acceptor framework comprises at least one amino acidsequence selected from the group consisting of: SEQ ID NO: 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and
 33. 5.The method of claim 4, wherein the antibody is a monoclonal antibody, achimeric antibody or a humanized antibody.
 6. A method of treatingretinal nerve fiber layer (RNFL) degeneration, comprising the step ofadministering to a mammal in need thereof an effective amount of acomposition comprising an isolated antibody, wherein the isolatedantibody comprises an antigen binding domain, said antibody capable ofbinding an epitope of a retinal guidance molecule (RGM), said antigenbinding domain comprising a heavy chain variable domain and a lightchain variable domain selected from the group consisting of: (a) theheavy chain variable domain having the amino acid sequence of SEQ IDNO:47 and the light chain variable domain having the amino acid sequenceof SEQ ID NO:44, (b) heavy chain variable domain having the amino acidsequence of SEQ ID NO:48 and the light chain variable domain having theamino acid sequence of SEQ ID NO:44, (c) the heavy chain variable domainhaving the amino acid sequence of SEQ ID NO:49 and the light chainvariable domain having the amino acid sequence of SEQ ID NO:44, (d) theheavy chain variable domain having the amino acid sequence of SEQ IDNO:50 and the light chain variable domain having the amino acid sequenceof SEQ ID NO:44, (e) the heavy chain variable domain having the aminoacid sequence of SEQ ID NO:47 and the light chain variable domain havingthe amino acid sequence of SEQ ID NO:45, (f) the heavy chain variabledomain having the amino acid sequence of SEQ ID NO:48 and the lightchain variable domain having the amino acid sequence of SEQ ID NO:45,(g) the heavy chain variable domain having the amino acid sequence ofSEQ ID NO:49 and the light chain variable domain having the amino acidsequence of SEQ ID NO:45, (h) the heavy chain variable domain having theamino acid sequence of SEQ ID NO:50 and the light chain variable domainhaving the amino acid sequence of SEQ ID NO:45, (i) the heavy chainvariable domain having the amino acid sequence of SEQ ID NO:47 and thelight chain variable domain having the amino acid sequence of SEQ IDNO:46, (j) the heavy chain variable domain having the amino acidsequence of SEQ ID NO:48 and the light chain variable domain having theamino acid sequence of SEQ ID NO:46, (k) the heavy chain variable domainhaving the amino acid sequence of SEQ ID NO:49 and the light chainvariable domain having the amino acid sequence of SEQ ID NO:46, (l) theheavy chain variable domain having the amino acid sequence of SEQ IDNO:50 and the light chain variable domain having the amino acid sequenceof SEQ ID NO:46, (m) the heavy chain variable domain having the aminoacid sequence of SEQ ID NO:48 and the light chain variable domain havingthe amino acid sequence of SEQ ID NO:51, (n) the heavy chain variabledomain having the amino acid sequence of SEQ ID NO:48 and the lightchain variable domain having the amino acid sequence of SEQ ID NO:52,(o) the heavy chain variable domain having the amino acid sequence ofSEQ ID NO:48 and the light chain variable domain having the amino acidsequence of SEQ ID NO:53, (p) the heavy chain variable domain having theamino acid sequence of SEQ ID NO:48 and the light chain variable domainhaving the amino acid sequence of SEQ ID NO:54, (q) the heavy chainvariable domain having the amino acid sequence of SEQ ID NO:50 and thelight chain variable domain having the amino acid sequence of SEQ IDNO:51, (r) the heavy chain variable domain having the amino acidsequence of SEQ ID NO:50 and the light chain variable domain having theamino acid sequence of SEQ ID NO:52, (s) the heavy chain variable domainhaving the amino acid sequence of SEQ ID NO:50 and the light chainvariable domain having the amino acid sequence of SEQ ID NO:53, and (t)the heavy chain variable domain having the amino acid sequence of SEQ IDNO:50 and the light chain variable domain having the amino acid sequenceof SEQ ID NO:54.
 7. The method of claim 6, wherein said treatment is antherapeutic, neuroregenerative or neuroprotective, local or systemictreatment.
 8. The method of claim 6, wherein the antibody is amonoclonal antibody, a chimeric antibody or a humanized antibody.